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[3H]Nociceptin ligand-binding and nociceptin opioid receptor mRNA expression in the human brain
Abstract
Following the cloning of the novel nociceptin opioid receptor (NOP(1)) and the identification of its endogenous ligand orphanin FQ/nociceptin the distribution and functional role of the NOP(1) receptor system have been studied mainly in the rodent CNS. In the present study the regional distribution and splice variant expression of the NOP(1) receptor was investigated in the adult human brain using [(3)H]-nociceptin autoradiography, NOP(1) reverse transcriptase PCR and mRNA in situ hybridization. Ligand binding revealed strong expression of functional NOP(1) receptors in the cerebral cortex and moderate signals in hippocampus and cerebellum. Interestingly, the NOP(1) receptor specific ligand was also strongly bound in the human striatum. A matching pattern of mRNA expression was observed with high amounts of NOP(1) mRNA in the prefrontal and cingulate cortex as well as in the dentate gyrus of the hippocampus. mRNA levels in the Ammon's horn and cerebellar cortex were moderate and low in the striatum. A considerable expression of N-terminal NOP(1) splice variant mRNAs was not detectable in the human brain by means of in situ hybridization. This suggests that functional NOP(1) receptors in the human brain are encoded by N-terminal full length NOP(1) transcripts. The present data on the anatomical distribution of nociceptin binding sites and NOP(1) receptor mRNA contribute to the knowledge about opioid receptor systems in the human brain and may promote the understanding of function and pharmacology of the orphanin FQ/nociceptin receptor system in the human CNS.
... The nucleotide sequence and amino acid sequence of the NOP receptors from rhesus monkeys are 96-98% identical to human NOP receptors (Koga et al., 2009). Anatomical studies have indicated that the distribution of NOP receptors in the CNS of non-human primates is largely similar to those observed in humans (Peluso et al., 1998;Berthele et al., 2003;Bridge et al., 2003). Notable areas of species differences include lower expression levels in raphe nuclei in nonhuman primates, compared with rodents (Neal et al., 1999a,b;Berthele et al., 2003;Bridge et al., 2003), and higher expression levels in caudate nucleus and putamen in nonhuman primates and humans, but not in rodents (Peluso et al., 1998;Berthele et al., 2003;Bridge et al., 2003). ...
... Anatomical studies have indicated that the distribution of NOP receptors in the CNS of non-human primates is largely similar to those observed in humans (Peluso et al., 1998;Berthele et al., 2003;Bridge et al., 2003). Notable areas of species differences include lower expression levels in raphe nuclei in nonhuman primates, compared with rodents (Neal et al., 1999a,b;Berthele et al., 2003;Bridge et al., 2003), and higher expression levels in caudate nucleus and putamen in nonhuman primates and humans, but not in rodents (Peluso et al., 1998;Berthele et al., 2003;Bridge et al., 2003). However, there has been no neurobiological study of the functions of NOP receptors on nociceptive processes, particularly in the supraspinal neural circuits (Schröder et al., 2014). ...
... Anatomical studies have indicated that the distribution of NOP receptors in the CNS of non-human primates is largely similar to those observed in humans (Peluso et al., 1998;Berthele et al., 2003;Bridge et al., 2003). Notable areas of species differences include lower expression levels in raphe nuclei in nonhuman primates, compared with rodents (Neal et al., 1999a,b;Berthele et al., 2003;Bridge et al., 2003), and higher expression levels in caudate nucleus and putamen in nonhuman primates and humans, but not in rodents (Peluso et al., 1998;Berthele et al., 2003;Bridge et al., 2003). However, there has been no neurobiological study of the functions of NOP receptors on nociceptive processes, particularly in the supraspinal neural circuits (Schröder et al., 2014). ...
Background and Purpose
Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor agonists display a promising analgesic profile in preclinical studies. However, supraspinal N/OFQ produced hyperalgesia in rodents and such an effect has not been addressed in primates. Thus, the aim of this study was to investigate the effects of centrally administered ligands on regulating pain and itch in nonhuman primates. In particular, nociceptive thresholds affected by intracisternal N/OFQ were compared to morphine and substance P, known to provide analgesia and mediate hyperalgesia, respectively, in humans.
Experimental Approach
An intrathecal catheterization was established to enable intracisternal and lumbar intrathecal administration in awake and unanesthetized rhesus monkeys. Nociceptive responses were measured by using the warm water tail-withdrawal assay. Itch scratching responses were scored from video tapes recording behavioral activities of monkeys in their home cages. Antagonist studies were conducted to validate the receptor mechanisms underlying intracisternally-elicited behavioral responses.
Key Results
Intracisternal morphine 100 nmol elicited more head scratches as compared to those of intrathecal morphine. Distinct dermatomal scratching locations between the two routes suggest a corresponding activation of supraspinal and spinal mu opioid receptors. Unlike intracisternal substance P-induced hyperalgesia, intracisternal N/OFQ 100 nmol produced NOP receptor-mediated antinociceptive effects; both peptides did not increase scratching responses.
Conclusions and Implications
Collectively, these results demonstrate differential actions of ligands in the primate supraspinal region in regulating pain and itch. This study not only improves scientific understanding of the N/OFQ-NOP receptor system in pain processing, but also supports the therapeutic potential of NOP-related ligands as analgesics.
... Brain radioactivity and kinetic analysis in human have detected low levels of NOP in the cerebellum (Lohith et al., 2012). PET tracing with nociceptin radioligands and in vitro methods were used to show that μ, κ and NOP receptors are expressed in the human cerebellum (Berthele et al., 2003;Lohith et al., 2012). Several studies have evaluated the binding of different ligands to the opioid receptors in the human cerebellum which has confirmed the presence of opioid receptors in the cerebellum (Berthele et al., 2005;Maarrawi et al., 2007;Naganawa et al., 2014;Zagon et al., 1990). ...
... The distribution of opioid receptors in extra-cerebellar brain regions. (Besse et al., 1990;Christie, 1991;Light et al., 2017;Puryear et al., 2020;Slamberová et al., 2003;Stein et al., 2003) Physical dependence Miosis Euphoria kappa (κ) + + + + + + + Immunoelectron microscopy Analgesia Gi (Hein et al., 2021;Johansson et al., 2001;Kitchen et al., 1997;Kreibich et al., 2008;Mansour et al., 1987;Puryear et al., 2020;Rácz and Halasy, 2003;Sirohi and Walker, 2015;Stein et al., 2003) Dissociative/ hallucinogenic miosis Dysphoria Neuroprotection Sedation Delta (δ) + + + + + + + Autoradiography Analgesia Gi (Bausch et al., 1995;Mansour et al., 1987;Olianas et al., 2012;Pan et al., 2002;Puryear et al., 2020;Stein et al., 2003) Physical dependence (Berthele et al., 2003;Fukuda et al., 1998;Neal et al., 1999;Puryear et al., 2020;Stein et al., 2003) Development of tolerance to μ-Opioid agonists ...
Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic (DAergic) neurons of the substantia nigra pars compacta (SNc) by neurodegeneration. Recent findings in animal models of PD propose tonic inhibition of the remaining DA neurons through GABA release from reactive glial cells. Movement dysfunctions could be ameliorated by promotion of activity in dormant DA cells. The endocannabinoid system (ECS) is extensively present in basal ganglia (BG) and is known as an indirect modulator of DAergic neurotransmission, thus drugs designed to target this system have shown promising therapeutic potential in PD patients. Interestingly, down/up-regulation of cannabinoid receptors (CBRs) varies across the different stages of PD, suggesting that some of the motor/ non-motor deficits may be related to changes in CBRs. Determination of the profile of changes of these receptors across the different stages of PD as well as their neural distribution within the BG could improve understanding of PD and identify pathways important in disease pathobiology. In this review, we focus on temporal and spatial alterations of CBRs during PD in the BG. At present, as inconclusive, but suggestive results have been obtained, future investigations should be conducted to extend preclinical studies examining CBRs changes within each stage in controlled clinical trials in order to determine the potential of targeting CBRs in management of PD.
... Brain radioactivity and kinetic analysis in human have detected low levels of NOP in the cerebellum (Lohith et al., 2012). PET tracing with nociceptin radioligands and in vitro methods were used to show that μ, κ and NOP receptors are expressed in the human cerebellum (Berthele et al., 2003;Lohith et al., 2012). Several studies have evaluated the binding of different ligands to the opioid receptors in the human cerebellum which has confirmed the presence of opioid receptors in the cerebellum (Berthele et al., 2005;Maarrawi et al., 2007;Naganawa et al., 2014;Zagon et al., 1990). ...
... The distribution of opioid receptors in extra-cerebellar brain regions. (Besse et al., 1990;Christie, 1991;Light et al., 2017;Puryear et al., 2020;Slamberová et al., 2003;Stein et al., 2003) Physical dependence Miosis Euphoria kappa (κ) + + + + + + + Immunoelectron microscopy Analgesia Gi (Hein et al., 2021;Johansson et al., 2001;Kitchen et al., 1997;Kreibich et al., 2008;Mansour et al., 1987;Puryear et al., 2020;Rácz and Halasy, 2003;Sirohi and Walker, 2015;Stein et al., 2003) Dissociative/ hallucinogenic miosis Dysphoria Neuroprotection Sedation Delta (δ) + + + + + + + Autoradiography Analgesia Gi (Bausch et al., 1995;Mansour et al., 1987;Olianas et al., 2012;Pan et al., 2002;Puryear et al., 2020;Stein et al., 2003) Physical dependence (Berthele et al., 2003;Fukuda et al., 1998;Neal et al., 1999;Puryear et al., 2020;Stein et al., 2003) Development of tolerance to μ-Opioid agonists ...
Increasing evidence suggests that the cerebellum could play a role in the higher cognitive processes involved in addiction as the cerebellum contains anatomical and functional pathways to circuitry controlling motivation and saliency. In addition, the cerebellum exhibits a widespread presence of receptors, including opioid receptors which are known to play a prominent role in synaptic and circuit mechanisms of plasticity associated with drug use and development of addiction to opioids and other drugs of abuse. Further, the presence of perineural nets (PNNs) in the cerebellum which contain proteins known to alter synaptic plasticity could contribute to addiction. The role the cerebellum plays in processes of addiction is likely complex, and could depend on the particular drug of abuse, the pattern of use, and the stage of the user within the addiction cycle. In this review, we discuss functional and structural modifications shown to be produced in the cerebellum by opioids that exhibit dependency-inducing properties which provide support for the conclusion that the cerebellum plays a role in addiction.
... These atomic details of ligand-receptor recognition explain marked differences in the binding selectivity of corresponding ligands in spite of high sequence homology between the NOP receptor and classical opioid receptors (Calo & Guerrini, 2013;Calo, Guerrini, Rizzi, Salvadori, & Regoli, 2000;Schr€ oder, Lambert, Ko, & Koch, 2014). Like N/OFQ, the NOP receptor is abundant in multiple brain areas and spinal cord (Berthele et al., 2003;Neal et al., 1999a), indicating that the N/OFQ-NOP receptor system plays a fundamental role in regulating several functions including pain. ...
... Unlike findings in rodents, intrathecal administration of N/OFQ over a wide range from femtomole to nanomole doses only produced antinociceptive effects without eliciting pronociceptive responses in a monkey thermal nociceptive assay, and these effects can be reversed by a NOP receptor antagonist (Ko & Naughton, 2009;Ko, Wei, Woods, & Kennedy, 2006). Although NOP receptors are expressed in neural substrates involved in pain processing in rodents and primates (Berthele et al., 2003;Bridge et al., 2003;Neal et al., 1999a), there is no anatomical study to directly compare the distribution of NOP receptors in different populations of neurons. It is important to further investigate the nature of nociceptive neurons expressing NOP receptors and determine if they have different properties for releasing pain-inhibiting and -eliciting neuropeptides between rodents and primates. ...
It has been two decades since the peptide, nociceptin/orphanin FQ (N/OFQ), and its cognate (NOP) receptor were discovered. Although NOP receptor activation causes a similar pattern of intracellular actions as mu opioid (MOP) receptors, NOP receptor-mediated pain modulation in rodents are more complicated than MOP receptor activation. In this review, we highlight the functional evidence of spinal, supraspinal, and systemic actions of NOP receptor agonists for regulating pain. In rodents, effects of the N/OFQ-NOP receptor system in spinal and supraspinal sites for modulating pain are bidirectional depending on the doses, assays, and pain modalities. The net effect of systemically administered NOP receptor agonists may depend on relative contribution of spinal and supraspinal actions of the N/OFQ-NOP receptor signaling in rodents under different pain states. In stark contrast, NOP receptor agonists produce only antinociception and antihypersensitivity in spinal and supraspinal regions of nonhuman primates regardless of doses and assays. More importantly, NOP receptor agonists and a few bifunctional NOP/MOP receptor agonists do not exhibit reinforcing effects (abuse liability), respiratory depression, itch pruritus, nor do they delay the gastrointestinal transit function (constipation) in nonhuman primates. Depending upon their intrinsic efficacies for activating NOP and MOP receptors, bifunctional NOP/MOP receptor agonists warrant additional investigation in primates regarding their side effect profiles. Nevertheless, NOP receptor-related agonists display a much wider therapeutic window as compared to that of MOP receptor agonists in primates. Both selective NOP receptor agonists and bifunctional NOP/MOP receptor agonists hold a great potential as effective and safe analgesics without typical opioid-associated side effects in humans.
... The NOP receptor is also localized in 5hydroxytryptaminergic, noradrenergic, and dopaminergic nuclei, such as the raphe complex, the locus coeruleus, the nucleus of the solitary tract, the ventral tegmental area, and the substantia nigra (Neal et al., 1999a;Mollereau and Mouledous, 2000). A similar pattern of N/OFQ and NOP receptor expression in human and rodent CNS has been observed (Peluso et al., 1998;Berthele et al., 2003;Witta et al., 2004). ...
... Moreover, investigating the effect of supraspinally delivered NOP receptor agonists on nociception in NHPs will further contribute to our understanding of species differences in antinociceptive efficacy of systemically administered NOP receptor agonists and is, therefore, highly warranted. Also in humans, both N/OFQ and NOP receptor mRNA and protein has been detected in nociceptive structures of the CNS and peripheral nervous system (Peluso et al., 1998;Mollereau and Mouledous, 2000;Berthele et al., 2003;Witta et al., 2004). The pattern of NOP receptor expression in humans was in general agreement with that seen in NHPs (Peluso et al., 1998;Bridge et al., 2003). ...
Despite high sequence similarity between NOP (nociceptin/orphaninFQ opioid peptide) and opioid receptors, marked differences in endogenous ligand selectivity, signal transduction, phosphorylation, desensitization, internalization and trafficking have been identified; underscoring the evolutionary difference between NOP and opioid receptors. Activation of NOP receptors affects nociceptive transmission in a site-specific manner, with antinociceptive effects prevailing after peripheral and spinal activation, and pronociceptive effects after supraspinal activation in rodents. The net effect of systemically administered NOP receptor agonists on nociception is proposed to depend on the relative contribution of peripheral, spinal and supraspinal activation and this may depend on experimental conditions. Functional expression and regulation of NOP receptors at peripheral and central sites of the nociceptive pathway exhibits a high degree of plasticity under conditions of neuropathic and inflammatory pain. In rodents, systemically administered NOP receptor agonists exerted antihypersensitive effects in models of neuropathic and inflammatory pain. However they were largely ineffective in acute pain while concomitantly evoking severe motor side effects. In contrast, systemic administration of NOP receptor agonists to non-human primates (NHPs) exerted potent and efficacious antinociception in the absence of motor and sedative side effects. The reason for this species difference with respect to antinociceptive efficacy and tolerability is not clear. Moreover, co-activation of NOP and μ-opioid peptide (MOP) receptors synergistically produced antinociception in NHPs. Hence, both selective NOP receptor as well as NOP/MOP receptor agonists may hold potential for clinical use as analgesics effective in conditions of acute and chronic pain.
... This suggests that up-regulation of DOR or KOR in cells expressing the NOP receptor would be one means of reducing N/OFQmediated calcium channel modulation. Although autoradiographic and/or in situ hybridization studies suggest that KOR and NOP receptors are regionally colocalized in the descending analgesic reward pathways (Mansour et al., 1995;Berthele et al., 2003) and that DOR and NOP receptors are regionally colocalized in the cortex, nucleus accumbens, amygdala, and dorsal horn (Neal et al., 1999;Berthele et al., 2003), these methods do not confirm colocalization at a cellular level. However, functional evidence also supports cellular colocalization of KOR/NOP receptor in PAG and RVM, suggesting that KOR/NOP receptor heterodimerization may mediate nociceptive processing (Vaughan et al., 2001(Vaughan et al., , 2003. ...
... This suggests that up-regulation of DOR or KOR in cells expressing the NOP receptor would be one means of reducing N/OFQmediated calcium channel modulation. Although autoradiographic and/or in situ hybridization studies suggest that KOR and NOP receptors are regionally colocalized in the descending analgesic reward pathways (Mansour et al., 1995;Berthele et al., 2003) and that DOR and NOP receptors are regionally colocalized in the cortex, nucleus accumbens, amygdala, and dorsal horn (Neal et al., 1999;Berthele et al., 2003), these methods do not confirm colocalization at a cellular level. However, functional evidence also supports cellular colocalization of KOR/NOP receptor in PAG and RVM, suggesting that KOR/NOP receptor heterodimerization may mediate nociceptive processing (Vaughan et al., 2001(Vaughan et al., , 2003. ...
The Nociceptin/Orphanin FQ (N/OFQ) peptide (NOP) receptor is the fourth and most recently discovered member of the opioid receptor superfamily that also includes μ, δ and κ opioid receptor subtypes (MOR, DOR and KOR, respectively). The widespread anatomical distribution of the NOP receptor enables the modulation of several physiological processes by its endogenous agonist, N/OFQ. Accordingly, the NOP receptor has gained a lot of attention as a potential target for the development of ligands with therapeutic utility in several pathophysiological states. NOP receptor activation frequently results in effects opposing classical opioid receptor action, therefore regulation of the NOP receptor and conditions affecting its modulatory tone are important to understand. Mounting evidence illustrates a heterologous interaction of the NOP receptor with other G protein-coupled receptors, including MOR, DOR and KOR, which may subsequently influence their function. Our focus in this review is to summarize and discuss the findings that delineate the cellular mechanisms of NOP receptor signaling and regulation as well as the regulation of other receptors by N/OFQ and the NOP receptor.
... NOP receptors and N/OFQ are particularly abundant in limbic regions and brain areas related to stress regulation in rodents and humans [30][31][32][33]. Accordingly, studies have associated the N/OFQ system with both the memory processes [34] and the stress response [29]. ...
Chronic stress causes cognitive deficits, such as impairments in episodic-like hippocampus-dependent memory. Stress regulates an opioid-related neuropeptide named Nociceptin/Orphanin FQ (N/OFQ), the ligand of the G protein-coupled receptor NOP. Since this peptide has deleterious effects on memory, we hypothesized that the N/OFQ system could be a mediator of the negative effects of stress on memory. Chronic stress was mimicked by chronic exposure to corticosterone (CORT). The NOP receptor was either acutely blocked using selective antagonists, or knocked-down specifically in the hippocampus using genetic tools. Long-term memory was assessed in the object recognition (OR) and object location (OL) paradigms. Acute injection of NOP antagonists before learning had a negative impact on memory in naive mice whereas it restored memory performances in the chronic stress model. This rescue was associated with a normalization of neuronal cell activity in the CA3 part of the hippocampus. Chronic CORT induced an upregulation of the N/OFQ precursor in the hippocampus. Knock-down of the NOP receptor in the CA3/Dentate Gyrus region prevented memory deficits in the CORT model. These data demonstrate that blocking the N/OFQ system can be beneficial for long-term memory in a neuroendocrine model of chronic stress. We therefore suggest that NOP antagonists could be useful for the treatment of memory deficits in stress-related disorders.
... NOPR is abundantly expressed within the central nervous system [6][7][8] , in line with the broad range of neural and cognitive functions regulated by this endogenous opioid system 9, 10 . In particular, NOPR and preproN/OFQ (PNOC)-expressing neurons are highly enriched in the ventral tegmental area (VTA), arcuate nucleus of the hypothalamus (ARC), dorsal striatum, nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) 7 . ...
Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.
... Human MOP and NOP receptors are coexpressed in the human brain, especially in laminae I~III of the prefrontal cortex, laminae II~V of the cingulate cortex, and the caudate nucleus of the basal ganglia [4][5][6]. MOP and NOP receptors are natively coexpressed in ...
We attempted to examine the alterations elicited by opioids via coexpressed μ-opioid (MOP) and nociceptin/orphanin FQ (NOP) receptors for receptor localization and Erk1/2 (p44/42 MAPK) in human embryonic kidney (HEK) 293 cells. Through two-photon microscopy, the proximity of MOP and NOP receptors was verified by fluorescence resonance energy transfer (FRET), and morphine but not buprenorphine facilitated the process of MOP-NOP heterodimerization. Single-particle tracking (SPT) further revealed that morphine or buprenorphine hindered the movement of the MOP-NOP heterodimers. After exposure to morphine or buprenorphine, receptor localization on lipid rafts was detected by immunocytochemistry, and phosphorylation of Erk1/2 was determined by immunoblotting in HEK 293 cells expressing MOP, NOP, or MOP+NOP receptors. Colocalization of MOP and NOP on lipid rafts was enhanced by morphine but not buprenorphine. Morphine stimulated the phosphorylation of Erk1/2 with a similar potency in HEK 293 cells expressing MOP and MOP+NOP receptors, but buprenorphine appeared to activate Erk1/2 solely through NOP receptors. Our results suggest that opioids can fine-tune the cellular localization of opioid receptors and phosphorylation of Erk1/2 in MOP+NOP-expressing cells.
... Genetic deletion of NOPR does not alter acute pain sensitivity but exacerbates hyperalgesic responses in conditions of persistent inflammatory pain (Depner et al., 2003;Rizzi et al., 2011). However, significant differences in NOPR supraspinal distribution and localization is observed between species, particularly between preclinical animal models and non-human primates/humans (Florin et al., 2000;Berthele et al., 2003). As such, the effects of NOPR manipulations in preclinical models of pain may not directly translate to clinical populations (Spetea et al., 2022). ...
Decades of research advances have established a central role for endogenous opioid systems in regulating reward processing, mood, motivation, learning and memory, gastrointestinal function, and pain relief. Endogenous opioid systems are present ubiquitously throughout the central and peripheral nervous system. They are composed of four families, namely the μ (MOPR), κ (KOPR), δ (DOPR), and nociceptin/orphanin FQ (NOPR) opioid receptors systems. These receptors signal through the action of their endogenous opioid peptides β-endorphins, dynorphins, enkephalins, and nociceptins, respectfully, to maintain homeostasis under normal physiological states. Due to their prominent role in pain regulation, exogenous opioids—primarily targeting the MOPR, have been historically used in medicine as analgesics, but their ability to produce euphoric effects also present high risks for abuse. The ability of pain and opioid use to perturb endogenous opioid system function, particularly within the central nervous system, may increase the likelihood of developing opioid use disorder (OUD). Today, the opioid crisis represents a major social, economic, and public health concern. In this review, we summarize the current state of the literature on the function, expression, pharmacology, and regulation of endogenous opioid systems in pain. Additionally, we discuss the adaptations in the endogenous opioid systems upon use of exogenous opioids which contribute to the development of OUD. Finally, we describe the intricate relationship between pain, endogenous opioid systems, and the proclivity for opioid misuse, as well as potential advances in generating safer and more efficient pain therapies.
... Interestingly, the NOP receptor and its endogenous ligand, nociceptin, have also been implicated in psychiatric disease (Post et al., 2016;Khan et al., 2018). In adults, the receptor is highly expressed in brain regions associated with mental illness, including the cortex, hippocampus, amygdala, thalamus, hypothalamus, and dorsal raphe (Neal et al., 2001;Berthele et al., 2003;Lambert, 2008;Kimura et al., 2011;Lohith et al., 2012). Further, nociceptin levels are elevated in patients suffering from bipolar disorder and major depression (Wang et al., 2009). ...
The misuse of opioids has reached epidemic proportions over the last decade, with over 2.1 million people in the United States suffering from substance use disorders related to prescription opioid pain relievers. This increase in opioid misuse affects all demographics of society, including women of child-bearing age, which has led to a rise in opioid use during pregnancy. Opioid use during pregnancy has been associated with increased risk of obstetric complications and adverse neonatal outcomes, including neonatal abstinence syndrome. Currently, opioid use disorder in pregnant women is treated with long-acting opioid agonists, including buprenorphine. Although buprenorphine reduces illicit opioid use during pregnancy and improves infant outcomes at birth, few long-term studies of the neurodevelopmental consequences have been conducted. The goal of the current experiments was to examine the effects of buprenorphine on the development of the cortex using fetal brain tissue, 3D brain cultures, and rodent models. First, we demonstrated that we can grow cortical and subpallial spheroids, which model the cellular diversity, connectivity, and activity of the developing human brain. Next, we show that cells in the developing human cortex express the nociceptin opioid (NOP) receptor and that buprenorphine can signal through this receptor in cortical spheroids. Using subpallial spheroids to grow inhibitory interneurons, we show that buprenorphine can alter interneuron development and migration into the cortex. Finally, using a rodent model of prenatal buprenorphine exposure, we demonstrate that alterations in interneuron distribution can persist into adulthood. Together, these results suggest that more research is needed into the long-lasting consequences of buprenorphine exposure on the developing human brain.
... The pharmacological profile of NOP is quite unique, despite close similarities of this receptor with the other three opioid receptors in terms of structure and signaling mechanisms. [17] NOP is present in many brain areas and in the spinal cord, [28] but also in peripheral tissues and in the immune system. [29] In the central Mr. Karol Wtorek received his M.Sci. ...
Nociceptin receptor (NOP) belongs to the family of opioid receptors but was discovered and characterized much later than the so called classical opioid receptors, μ, δ and κ (or MOP, DOP and KOP, resp.). Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of this receptor and it controls numerous important functions in the central nervous system and in the periphery, so its analogs may be developed as innovative drugs for the treatment of a variety of conditions and pathological states. Availability of potent and selective ligands with high affinity to NOP receptor is essential to fully understand the role of NOP‐N/OFQ system in the body, which in turn may lead to designing novel therapeutics. Here, we have focused on reviewing the structure of potent peptide‐based agonists, antagonists, biased analogs and bivalent ligands that target NOP receptor.
... Most of the information pertaining to the pharmacological action and function of N/OFQ in disease states has come from rodent studies, but emerging clinical findings support the hypothesis that increased levels of N/OFQ may modulate negative emotional states. Significant species differences in N/OFQ expression, localization and density of NOPs are apparent across rodents (Florin, Leroux-Nicollet, Meunier, & Costentin, 1997;Florin, Meunier, & Costentin, 2000), primates (Bridge, Wainwright, Reilly, & Oliver, 2003;Kimura et al., 2011) and humans (Berthele et al., 2003;Lohith et al., 2012). These differences could lead to differential regulation of physiological responses by the N/OFQ NOP system across species. ...
Since the serendipitous discovery of the first class of modern antidepressants in the 1950's, all pharmacotherapies approved by the Food and Drug Administration for major depressive disorder (MDD)have shared a common mechanism of action, increased monoaminergic neurotransmission. Despite the widespread availability of antidepressants, as many as 50% of depressed patients are resistant to these conventional therapies. The significant length of time required to produce meaningful symptom relief with these medications, 4–6 weeks, indicates that other mechanisms are likely involved in the pathophysiology of depression which may yield more viable targets for drug development. For decades, no viable candidate target with a different mechanism of action to that of conventional therapies proved successful in clinical studies. Now several exciting avenues for drug development are under intense investigation. One of these emerging targets is modulation of endogenous opioid tone. This review will evaluate preclinical and clinical evidence pertaining to opioid dysregulation in depression, focusing on the role of the endogenous ligands endorphin, enkephalin, dynorphin, and nociceptin/orphanin FQ (N/OFQ)and their respective receptors, mu (MOR), delta (DOR), kappa (KOR), and the N/OFQ receptor (NOP)in mediating behaviors relevant to depression and anxiety. Finally, putative opioid based antidepressants that are under investigation in clinical trials, ALKS5461, JNJ-67953964 (formerly LY2456302 and CERC-501)and BTRX-246040 (formerly LY-2940094)will be discussed. This review will illustrate the potential therapeutic value of targeting opioid dysregulation in developing novel therapies for MDD.
... The localization of the nociceptin/orphanin FQ peptide NOP receptor (N/OFQ-NOP receptor) system along with the activity of compounds in preclinical models served as an initial guide for clinical development of NOP receptor modulators. NOP receptors are broadly expressed in cortical regions, including the prefrontal and cingulate cortices, as well in the hippocampus and striatum (Berthele et al. 2003). This expression pattern positions N/OFQ to interact with multiple neural circuits that regulate mood, learning, and motor control (Zaveri 2016;Witkin et al. 2014). ...
Conventional antidepressants increase the efflux of biogenic amine neurotransmitters (the monoamine hypothesis of depression) in the central nervous system (CNS) and are the principle drugs used to treat major depressive disorder (MDD). However, the lack of efficacy in some patients, the slow onset of action, and the side effect profiles of existing antidepressants necessitate the exploration of additional treatment options. The discovery of the nociceptin/orphanin FQ peptide NOP receptor (N/OFQ-NOP receptor) system and its characterization in preclinical biological and pharmacological stress-related conditions supports the potential antidepressant and anti-stress properties of a NOP receptor antagonist for the treatment of neurobehavioral disorders. BTRX-246040 (formerly LY2940094) was designed to test this hypothesis in the clinic. A small clinical proof of concept study demonstrated efficacy of BTRX-246040 in MDD patients. In this study, BTRX-246040 (40 mg, p.o.) significantly reduced negative bias as assessed by the facial recognition test within 1 week of treatment and decreased depression symptoms after 8 weeks. BTRX-246040 also reduced depression symptoms in a second trial with heavy alcohol drinkers. Given the comorbidity of MDD and alcohol use disorder, a compound with such effects in patients could be a valuable addition to the medications available. A proof of concept study showed efficacy of BTRX-246040 in reducing heavy drinking and increasing the probability of abstinence in individuals diagnosed with alcohol dependence. In addition, plasma levels of gamma-glutamyl transferase were decreased by BTRX-246040 compared to placebo control implying improvement in liver function. Collectively, the clinical data reviewed within this chapter suggest that BTRX-264040 functions to normalize dysfunction in reward circuits. The overall efficacy and safety of this compound with a novel mechanism of action are encouraging of further clinical development. BTRX-246040 is currently under development for MDD by BlackThorn Therapeutics.
... Néanmoins, des études d'imagerie récentes, utilisant la technique de tomographie par émission de positons, rapportent une liaison de la nociceptine radioactive dans le cervelet équivalente à celle observée dans le thalamus chez le singe rhésus(Kimura et al. 2011 ; Fig. 12) et chez l'Homme(Lohith et al. 2012). Dans le cervelet humain adulte, le ligand et son récepteur sont en particulier exprimés dans le cortex cérébelleux(Berthele et al. 2003). ...
Le cervelet est une structure cérébrale impliquée dans de multiples fonctions motrices mais aussi cognitives et dont le développement postnatal est sous le contrôle de divers types de facteurs dont les neuropeptides. Les peptides capables d’agir sur le développement du cortex cérébelleux présentent généralement un profil d’expression particulier, avec chez le rongeur un pic d’expression au cours des 2 premières semaines postnatales. L’objectif de cette étude était d’identifier d’autres peptides présentant ce même type d’expression et de caractériser leurs potentiels effets au cours du développement du cortex cérébelleux, et plus particulièrement dans la mise en place des neurones en grain qui sont les plus abondants de cette structure. Pour cela, des cervelets de rats âgés de 8 à 90 jours ont été analysés par spectrométrie de masse. Parmi les 33 peptides identifiés, 4 présentent le profil recherché et nous avons choisi d’étudier l’un d’entre eux, la nociceptine. La mesure de l’expression du gène de la nociceptine et de son récepteur montre un profil d’expression similaire à celui observé en peptidomique. De plus, ces 2 gènes sont retrouvés principalement exprimés dans la couche granulaire interne du cortex cérébelleux par microdissection et qPcr. La recherche de la fonction de la nociceptine montre qu’elle exerce un effet neurotrophique en augmentant la survie et la différenciation des neurones en grain, sans affecter la motilité de ces cellules. Des tests préliminaires réalisés in vivo indiquent que la nociceptine est aussi capable de bloquer la toxicité induite par l’alcool. La dernière partie de l’étude avait pour but d’identifier de nouveaux neuropeptides exprimés dans le cervelet en utilisant une approche par séquençage de novo. L’application de filtres comme la récurrence des séquences peptidiques ou leur régulation au cours du développement a permis de ne retenir que 6 séquences pour la suite de l’analyse. Des études génomiques permettront de restreindre encore ce nombre afin de focaliser les tests d’activité biologique sur la ou les cibles qui ont la plus grande probabilité de correspondre à des peptides biologiquement actifs.
... The endogenous peptide, now known as nociceptin/orphanin FQ (N/OFQ), was identified in 1995, and is a heptadecapeptide with pro-nociceptive properties 9,10 . In vitro receptor autoradiography in rats and postmortem studies in humans have shown NOPr to be widely distributed, with greater density in cortical regions and the human striatum 11,12 . Positron emission tomography (PET) studies using the ligand [ 11 C]NOP-1A have corroborated these findings in vivo, with high concentrations of NOPr observed in the cerebral cortex and the striatum 13 . ...
Schizophrenia is a debilitating neuropsychiatric illness that is characterized by positive, negative, and cognitive symptoms. Research over the past two decades suggests that the nociceptin receptor system may be involved in domains affected in schizophrenia, based on evidence aligning it with hallmark features of the disorder. First, aberrant glutamatergic and striatal dopaminergic function are associated with psychotic symptoms, and the nociceptin receptor system has been shown to regulate dopamine and glutamate transmission. Second, stress is a critical risk factor for first break and relapse in schizophrenia, and evidence suggests that the nociceptin receptor system is also directly involved in stress modulation. Third, cognitive deficits are prevalent in schizophrenia, and the nociceptin receptor system has significant impact on learning and working memory. Last, reward processing is disrupted in schizophrenia, and nociceptin signaling has been shown to regulate reward cue salience. These findings provide the foundation for the involvement of the nociceptin receptor system in the pathophysiology of schizophrenia and outline the need for future research into this system.
... [ 11 C]NOP-1A binds with relatively high affinity to NOP receptors (equilibrium dissociation constant [K D ] = 0.15 nM). [ 11 C]NOP-1A total uptake in various brain regions (distribution volume [V T ]) is consistent with the known distribution of NOP receptors in human and primate brains (relatively high density in amygdala, striatum, and cerebral cortex; moderate density in cerebellum and midbrain) (14)(15)(16)(17). Blocking studies in primates with the specific NOP receptor antagonist SB-612111 (K D = 0.33 nM, 1.0 mg/kg intravenously) indicate that 50% to 70% of [ 11 C]NOP-1A V T in brain regions represents specific binding to NOP receptors (16). ...
Background
The neuropeptide transmitter nociceptin, which binds to the nociceptin/orphanin FQ peptide (NOP) receptor, is a core component of the brain's anti-stress system. Nociceptin exerts its anti-stress effect by counteracting the functions of corticotropin releasing factor, the primary stress-mediating neuropeptide in the brain. Basic investigations support a role for medications that target nociceptin receptors in the treatment of alcohol use disorders. Thus, it is of high interest to measure the in vivo status of NOP receptors in individuals with alcohol use disorders.
Methods
Here, we used [¹¹C]NOP-1A and PET to measure the in vivo binding to NOP receptors in 15 DSM-IV alcohol-dependent humans and 15 healthy controls matched for age, gender, and smoking status. Alcohol-dependent individuals with no comorbid psychiatric, medical, or drug abuse disorders were scanned following two weeks of outpatient monitored abstinence (confirmed with 3x/week urine alcohol metabolite testing). [¹¹C]NOP-1A distribution volume (VT) in regions of interest (including the amygdala, hippocampus, midbrain, striatal, and prefrontal cortical subdivisions) were measured with kinetic analysis using the arterial input function.
Results
Regional [¹¹C]NOP-1A VT in alcohol-dependence was not significantly different compared to healthy controls. No relationship between [¹¹C]NOP-1A VT and other clinical measures (including duration and severity of alcohol abuse, craving, anxiety or depressive symptoms) were significant after correction for the multiple hypotheses tested.
Conclusions
The results of this study do not support alterations in the binding to NOP receptors in alcohol dependence. However, this finding does not necessarily rule out alterations in nociceptin transmission in alcohol dependence.
... Brain autoradiography studies in rodents have revealed a very high concentration of NOP receptors in the frontal cortex, and there are particularly high levels of NOP receptors in the cingulate cortex (Neal et al. 1999;Sim and Childers 1997;Sim et al. 1996). Consistent with these rodent findings, human positron emission tomography (PET) and post-mortem autoradiography studies have also reported high distribution of NOP receptors in the cingulate cortex and striatum (Berthele et al. 2003;Lohith et al. 2014;Lohith et al. 2012). Moreover, NOP receptors are localized on dopaminergic nuclei in the VTA, and administration of a NOP receptor agonist inhibited dopamine neurotransmission in the VTA and NAc (Koizumi et al. 2004a;Koizumi et al. 2004b;Murphy et al. 1996;Murphy and Maidment 1999;Murphy et al. 2004). ...
RationaleMood disorders can be triggered by stress and are characterized by deficits in reward processing, including disrupted reward learning (the ability to modulate behavior according to past rewards). Reward learning is regulated by the anterior cingulate cortex (ACC) and striatal circuits, both of which are implicated in the pathophysiology of mood disorders. Objectives
Here, we assessed in rats the effects of a potent stressor (social defeat) on reward learning and gene expression in the ACC, ventral tegmental area (VTA), and striatum. Methods
Adult male Wistar rats were trained on an operant probabilistic reward task (PRT) and then exposed to 3 days of social defeat before assessment of reward learning. After testing, the ACC, VTA, and striatum were dissected, and expression of genes previously implicated in stress was assessed. ResultSocial defeat blunted reward learning (manifested as reduced response bias toward a more frequently rewarded stimulus) and was associated with increased nociceptin/orphanin FQ (N/OFQ) peptide mRNA levels in the striatum and decreased Fos mRNA levels in the VTA. Moreover, N/OFQ peptide and nociceptin receptor mRNA levels in the ACC, VTA and striatum were inversely related to reward learning. Conclusions
The behavioral findings parallel previous data in humans, suggesting that stress similarly disrupts reward learning in both species. Increased striatal N/OFQ mRNA in stressed rats characterized by impaired reward learning is consistent with accumulating evidence that antagonism of nociceptin receptors, which bind N/OFQ, has antidepressant-like effects. These results raise the possibility that nociceptin systems represent a molecular substrate through which stress produces reward learning deficits in mood disorders.
... We have recently discovered both labeled and unlabeled NOP receptor tracers with subnanomolar binding affinities with no intrinsic activity (i.e., antagonists), high selectivity, CNS penetration, and low nonspecific binding (Pedregal et al., 2012). 5 was safe, with favorable kinetics and reproducibility (Lohith et al., 2012(Lohith et al., , 2014, and exhibited distribution volume (V T ) consistent with known NOP density distribution (Berthele et al., 2003). ...
Therapeutic benefits from nociceptin opioid peptide receptor (NOP) antagonism were proposed for obesity, eating disorders, and depression. LY2940094 is a novel, orally bioavailable, potent, and selective NOP antagonist. We studied NOP receptor occupancy (RO) after single oral LY2940094 doses in rat hypothalamus and human brain with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LSN2810397) and positron emission tomography (PET) ([11C]NOP-1A) tracers, respectively. A bolus plus constant infusion tracer protocol with PET was employed in humans at 2.5 and 26.5 hours post-LY2940094 dose. RO was calculated from the change in regional distributional volume (VT) corrected for nondisplaceable volume using Lasson plots. RO followed a simple Emax relationship to plasma LY2940094 concentration reaching near complete occupancy in both species. For rat hypothalamus, plasma concentration at half-maximum RO (EC50) was 5.8 ng/mL. In humans, LY2940094 was well-tolerated and safe over the 4-40 mg dose range, and peaked in plasma at 2-6 hours after 1-2 hour lag, with approximate dose-proportional exposure. Following 4-40 mg doses, NOP RO was similar across prefrontal cortex, occipital cortex, putamen, and thalamus, with EC50 of 2.94 to 3.46 ng/mL, less than 2-fold lower than rat. Over 4-40 mg doses, LY2940094 mean plasma levels at peak and 24 hours were 7.93-102 and 1.17-14.1 ng/mL, corresponding to cross-region average NOP RO of 73-97% and 28-82%, respectively. Rat EC50 translates well to humans. LY2940094 readily penetrates human brain, and a once-daily oral dose of 40 mg achieves sustainably high (>80%) NOP RO levels suitable for testing clinical efficacy.
... Specificity controls for immunostaining were satisfactory: nerve structures were immunostained in a titre-specific manner ( Fig. 2A), and no immunostaining was observed when the primary antibody was omitted. Additionally, no staining was observed in the human cerebellum (Fig. 2B), a tissue in which absence or only very limited presence of both NOP receptor transcripts and N/OFQ binding has been demonstrated, 11,44,57 whereas neurofilament cocktail antibodies showed positive neuronal cell bodies and nerve fibres in serial sections. Similar immunoblotting results were reported with this antibody compared with other antibodies directed against the C or N terminus of the receptor. ...
The Nociceptin/Orphanin FQ peptide receptor (NOP), activated by its endogenous peptide ligand Nociceptin/Orphanin FQ (N/OFQ), exerts several effects including modulation of pain signalling. We have examined, for the first time, the tissue distribution of the NOP receptor in clinical visceral and somatic pain disorders by immunohistochemistry, and assessed functional effects of NOP and µ opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder sub-urothelium revealed a remarkable several-fold increase in Detrusor Overactivity (p<0.0001) and Painful Bladder Syndrome patient specimens (p=0.0014), compared to controls. In post-mortem control human DRGs, 75-80% of small/medium neurons (≤50 µm diameter) in the lumbar (somatic) and sacral (visceral) DRG were positive for NOP, and fewer large neurons; avulsion-injured cervical human DRG neurons showed similar numbers. NOP-immunoreactivity was significantly decreased in injured peripheral nerves (p=0.0004), and also in painful neuromas (p=0.025). Calcium imaging studies in cultured rat DRG neurons demonstrated dose-dependent inhibition of capsaicin responses in the presence of N/OFQ, with an IC50 of 8.6 pM. In cultured human DRG neurons, 32% inhibition of capsaicin responses was observed in the presence of 1 pM N/OFQ (p<0.001). The maximum inhibition of capsaicin responses was greater with N/OFQ than μ-opioid receptor agonist DAMGO. Our findings highlight the potential of NOP agonists, particularly in urinary bladder overactivity and pain syndromes. The regulation of NOP expression in visceral and somatic sensory neurons by target-derived neurotrophic factors deserves further study, and the efficacy of NOP selective agonists in clinical trials.
... In that study, three different splice variants were detected in rat brain tissue. However, a later study assessing human tissue reported no evidence for expression of alternative splice variants in human brain tissue, the ultimate target of interest (Berthele et al., 2003). Thus, even if splice variants existed in the rodent, yielding differential pharmacology with different ligands, it is unlikely that would translate into observable differences in human pharmacology. ...
Background:
The nociceptin/orphanin-FQ (or opioid receptor-like [ORL1]) receptor (NOP) is localized in the mesolimbic reward pathway and has been suggested to play a role in feeding, mood, stress, and addiction. Since its deorphanization in 1995, there has been a clear dichotomy in the literature regarding whether an agonist or antagonist would provide therapeutic benefit. Specifically, the literature reports indicate that NOP receptor antagonists produce efficacy in animal models of hyperphagia and antidepressant-like activity, whereas NOP agonists produce anxiolytic-like effects and dampen reward/addiction behaviors including ethanol consumption.
Methods:
We characterize here the potent, orally bioavailable NOP antagonist, LY2940094, in rodent models of ethanol consumption, including ethanol self-administration, progressive ratio operant self-administration, stress-induced reinstatement of ethanol seeking, and in vivo microdialysis in the nucleus accumbens.
Results:
LY2940094 dose dependently reduced homecage ethanol self-administration in Indiana alcohol-preferring (P) and Marchigian Sardinian alcohol-preferring (msP) rats, without affecting food/water intake or locomotor activity. Reduced ethanol intake in P rats did not show significant tolerance over 4 days of subchronic dosing. LY2940094 attenuated progressive ratio operant responding and break points for ethanol in P rats. Moreover, stress-induced reinstatement of ethanol seeking in msP rats was completely blocked by LY2940094. Furthermore, LY2940094 blocked ethanol-stimulated dopamine release in response to ethanol challenge (1.1 g/kg, intraperitoneally).
Conclusions:
Our findings demonstrate for the first time that blockade of NOP receptors attenuates ethanol self-administration and ethanol-motivated behaviors, stress-induced ethanol seeking, and ethanol-induced stimulation of brain reward pathways in lines of rats that exhibit excessive ethanol consumption. Results suggest that LY2940094 may have potential therapeutic utility in treating alcohol addiction.
... These effects were antagonized by a NOP receptor antagonist, confirming a striatal function for NOP receptors in the rat. Higher levels of NOP receptor expression are reported in the primate striatum (Berthele et al., 2003; Bridge et al., 2003). The functional roles of the N/OFQ-NOP receptor system in the striatum are not fully elucidated, but activation of NOP receptor has been shown to reduce the dyskinesias induced by chronic L-DOPA administration in experimental models of PD in both rats and nonhuman primates (Marti et al., 2012). ...
The NOP receptor (nociceptin/orphanin FQ opioid peptide receptor) is the most recently discovered member of the opioid receptor family and, together with its endogenous ligand, N/OFQ, make up the fourth members of the opioid receptor and opioid peptide family. Because of its more recent discovery, an understanding of the cellular and behavioral actions induced by NOP receptor activation are less well developed than for the other members of the opioid receptor family. All of these factors are important because NOP receptor activation has a clear modulatory role on mu opioid receptor-mediated actions and thereby affects opioid analgesia, tolerance development, and reward. In addition to opioid modulatory actions, NOP receptor activation has important effects on motor function and other physiologic processes. This review discusses how NOP pharmacology intersects, contrasts, and interacts with the mu opioid receptor in terms of tertiary structure and mechanism of receptor activation; location of receptors in the central nervous system; mechanisms of desensitization and downregulation; cellular actions; intracellular signal transduction pathways; and behavioral actions with respect to analgesia, tolerance, dependence, and reward. This is followed by a discussion of the agonists and antagonists that have most contributed to our current knowledge. Because NOP receptors are highly expressed in brain and spinal cord and NOP receptor activation sometimes synergizes with mu receptor-mediated actions and sometimes opposes them, an understanding of NOP receptor pharmacology in the context of these interactions with the opioid receptors will be crucial to the development of novel therapeutics that engage the NOP receptor.
... A caveat that is unfolding as the NOP receptor is being vetted as a human clinical target is that there are significant differences in the pharmacological effects of NOP ligands in rodents, compared to that in nonhuman primates and possibly humans, that need to be carefully considered and which impact the success of the preclinical advancement of suitable drug candidates and selection of clinical endpoints. Studies on localization of the NOP system in the rodent, 31, 32 primate 33,34 and human brain 35,36 suggests that there are important differences in the distribution and localization of the NOP-N/OFQ system between species, that may play a significant role in translating the validation of this system as a potential target for different pathophysiological states. ...
In the two decades since the discovery of the nociceptin opioid receptor (NOP) and its ligand, nociceptin/orphaninFQ (N/OFQ), steady progress has been achieved in understanding the pharmacology of this fourth opioid receptor/peptide system, aided by genetic and pharmacologic approaches. This research spawned an explosion of small-molecule NOP receptor ligands from discovery programs in major pharmaceutical companies. NOP agonists have been investigated for their efficacy in preclinical models of anxiety, cough, substance abuse, pain (spinal and peripheral) and urinary incontinence, whereas NOP antagonists have been investigated for treatment of pain, depression and motor symptoms in Parkinson's disease. Translation of preclinical findings into the clinic is guided by PET and receptor occupancy studies, particularly for NOP antagonists. Recent progress in preclinical NOP research suggests that NOP agonists may have clinical utility for pain treatment and substance abuse pharmacotherapy. This review discusses the progress towards validating the NOP-N/OFQ system as a therapeutic target.
... Brain autoradiography studies in rodents have revealed a very high concentration of NOP receptors in the frontal cortex, and there are particularly high levels of NOP receptors in the cingulate cortex (Neal et al. 1999;Sim and Childers 1997;Sim et al. 1996). Consistent with these rodent findings, human positron emission tomography (PET) and post-mortem autoradiography studies have also reported high distribution of NOP receptors in the cingulate cortex and striatum (Berthele et al. 2003;Lohith et al. 2014;Lohith et al. 2012). Moreover, NOP receptors are localized on dopaminergic nuclei in the VTA, and administration of a NOP receptor agonist inhibited dopamine neurotransmission in the VTA and NAc (Koizumi et al. 2004a;Koizumi et al. 2004b;Murphy et al. 1996;Murphy and Maidment 1999;Murphy et al. 2004). ...
... In the future, it would be interesting to see the modulation of physiological responses in monkeys following supraspinal administration of NOP agonists in order to gain better understanding of how supraspinal activation of NOP may regulate antinociceptive effects after systemic or spinal administration. Anatomical studies reveal that there are differences between rodents and primates in terms of the distribution of N/OFQ and its receptors (112,113). For example, reasonable expression of NOP mRNA and NOP-radioligand binding was detected in striatum and cerebellar cortex of primates in contrast with lack of expression reported in rodents (114)(115)(116). ...
Most widely used analgesic drugs are mu-opioid receptor agonists such as morphine. However, analgesic value of these drugs is compromised due to unwanted side effects including respiratory depression, abuse liability, itch, and tolerance to analgesia. Nociceptin/orphanin FQ receptor (NOP) is emerging as a potential analgesic target without abuse liability. Analgesic properties of NOP-related agonists have been investigated in rodents and monkeys. In rodents, spinal injection of NOP agonists produces antinociception against diverse pain modalities and also potentiates morphine-induced antinociception. In monkeys, both spinal and systemic administration of NOP agonists produces morphine-comparable antinociceptive effects against acute nociception, capsaicin-induced allodynia, and carrageenan-induced hyperalgesia. More importantly, NOP agonists do not produce respiratory depression, itch scratching, and reinforcing effects at the antinociceptive doses. Interestingly, spinal or systemic administration of NOP agonists can potentiate mu-opioid receptor mediated antinociception and widen the therapeutic window in monkeys. Therefore, NOP agonists have a promising analgesic value when injected alone or in combination with mu opioid analgesics. These studies further support the therapeutic potential of NOP-related ligands including selective NOP agonists and bifunctional NOP/MOP agonists as effective analgesics in order to achieve strong pain relief without concerns over abuse and safety.
As clinical use of currently available opioid analgesics is often impeded by dose-limiting adverse effects, such as abuse liability and respiratory depression, new approaches have been pursued to develop safe, effective, and non-addictive pain medications. After the identification of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor more than 25 years ago, NOP receptor-related agonists have emerged as a promising target for developing novel and effective opioids that modulate the analgesic and addictive properties of mu-opioid peptide (MOP) receptor agonists. In this review, we highlight the effects of the NOP receptor-related agonists compared with those of MOP receptor agonists in experimental rodent and more translational non-human primate (NHP) models and the development status of key NOP receptor-related agonists as potential safe and non-addictive analgesics. Several lines of evidence demonstrated that peptidic and non-peptidic NOP receptor agonists produce potent analgesic effects by intrathecal delivery in NHPs. Moreover, mixed NOP/MOP receptor partial agonists (e.g., BU08028, BU10038, and AT-121) display potent analgesic effects when administered intrathecally or systemically, without eliciting adverse effects, such as respiratory depression, itch behavior, and signs of abuse liability. More importantly, cebranopadol, a mixed NOP/opioid receptor agonist with full efficacy at NOP and MOP receptors, produces robust analgesic efficacy with reduced adverse effects, conferring promising outcomes in clinical studies. A balanced coactivation of NOP and MOP receptors is a strategy that warrants further exploration and refinement for the development of novel analgesics with a safer and effective profile.
The misuse of opioids has reached epidemic proportions over the last decade, with over 2.1 million people in the U.S. suffering from substance use disorders related to prescription opioid pain relievers. This increase in opioid misuse affects all demographics of society, including women of child-bearing age, which has led to a rise in opioid use during pregnancy. Opioid use during pregnancy has been associated with increased risk of obstetric complications and adverse neonatal outcomes, including neonatal abstinence syndrome. Currently, opioid use disorder in pregnant women is treated with long-acting opioid agonists, including buprenorphine. Although buprenorphine reduces illicit opioid use during pregnancy and improves infant outcomes at birth, few long-term studies of the neurodevelopmental consequences have been conducted. The goal of the current experiments was to examine the effects of buprenorphine on the development of the cortex using fetal brain tissue, 3D brain cultures, and rodent models. First, we demonstrated that we can grow cortical and subpallial spheroids, which model the cellular diversity, connectivity, and activity of the developing human brain. Next, we show that cells in the developing human cortex express the nociceptin opioid (NOP) receptor and that buprenorphine can signal through this receptor in cortical spheroids. Using subpallial spheroids to grow inhibitory interneurons, we show that buprenorphine can alter interneuron development and migration into the cortex. Finally, using a rodent model of prenatal buprenorphine exposure, we demonstrate that alterations in interneuron distribution can persist into adulthood. Together, these results suggest that more research is needed into the long-lasting consequences of buprenorphine exposure on the developing human brain.
Rationale and Objective
One objective of the National Institutes of Health Helping to End Addiction Long-term (HEAL) initiative is to accelerate research on safer and more effective medications for both pain and opioid use disorder. Ligands that activate the nociceptin opioid peptide receptor (NOP) constitute one class of candidate drugs for both applications. The present preclinical study determined the effectiveness of the NOP agonist Ro 64-6198 to produce antinociception in a pain-depressed behavior procedure and attenuate opioid self-administration in a heroin-vs-food choice procedure.
Methods
In Experiment 1, Adult Sprague-Dawley rats were equipped with microelectrodes and trained to respond for electrical brain stimulation in an intracranial self-stimulation (ICSS) procedure. The potency, time course, and receptor mechanism of effects produced by R0 64-6198 alone (0.32-3.2 mg/kg) on ICSS were examined, followed by evaluation of 0.32-1.0 mg/kg Ro 64-6198 effectiveness to block lactic acid-induced depression of ICSS. In Experiment 2, rats self-administered heroin under a heroin-vs-food choice procedure during a regimen of repeated, daily intraperitoneal administration of vehicle or Ro 64-6198 (1-3.2 mg/kg/day).
Results
Ro 64-6198 produced dose- and time-dependent ICSS depression that was blocked by the selective NOP antagonist SB612111 but not by naltrexone. Ro 64-6198 failed to block acid-induced depression of ICSS. Repeated Ro 64-6198 pretreatment also failed to attenuate heroin-vs-food choice up to doses that significantly decreased operant behavior.
Conclusions
These results do not support the utility of Ro 64-6198 as a stand-alone medication for either acute pain or opioid use disorder.
The neuropeptide nociceptin/orphanin FQ (N/OFQ) can be released by stressors and is associated with disorders of emotion regulation and reward processing. N/OFQ and its receptor, NOP, are enriched in dopaminergic pathways, and intra-ventricular agonist delivery decreases dopamine levels in the dorsal striatum, nucleus accumbens (NAc), and ventral tegmental area (VTA). We used whole-cell electrophysiology in acute rat midbrain slices to investigate synaptic actions of N/OFQ. N/OFQ was primarily inhibitory, causing outward currents in both immunocytochemically identified dopaminergic (tyrosine hydroxylase positive (TH(+))) and non-dopaminergic (TH(-)) VTA neurons; effect at 1 μm: 20 ± 4 pA. Surprisingly, this effect was mediated by augmentation of postsynaptic GABAAR currents, unlike the substantia nigra pars compacta (SNc), where the N/OFQ-induced outward currents were K+ channel dependent. A smaller population, 17% of all VTA neurons, responded to low concentrations of N/OFQ with inward currents (10 nm: -11 ± 2 pA). Following 100 nm N/OFQ, the response to a second N/OFQ application was markedly diminished in VTA neurons (14 ± 10% of first response) but not in SNc neurons (90 ± 20% of first response). N/OFQ generated outward currents in medial prefrontal cortex (mPFC)-projecting VTA neurons, but inward currents in a subset of posterior anterior cingulate cortex (pACC)-projecting VTA neurons. While N/OFQ inhibited NAc-projecting VTA cell bodies, it had little effect on electrically or optogenetically evoked terminal dopamine release in the NAc measured ex vivo with fast scan cyclic voltammetry (FSCV). These results extend our understanding of the N/OFQ system in brainstem circuits implicated in many neurobehavioral disorders.
Despite similar distribution patterns and intracellular events observed in the nociceptin/orphanin FQ peptide (NOP) receptor and other opioid receptors, NOP receptor activation displays unique pharmacological profiles. Several researchers have identified a variety of peptide and non-peptide ligands to determine the functional roles of NOP receptor activation and observed that NOP receptorrelated ligands exhibit pain modality-dependent pain processing. Importantly, NOP receptor activation results in anti-nociception and anti-hypersensitivity at the spinal and supraspinal levels regardless of the experimental settings in non-human primates (NHPs). Given that the NOP receptor agonists synergistically enhance mu-opioid peptide (MOP) receptor agonist-induced anti-nociception, it has been hypothesized that dual NOP and MOP receptor agonists may display promising functional properties as analgesics. Accumulating evidence indicates that the mixed NOP/opioid receptor agonists demonstrate favorable functional profiles. In NHP studies, bifunctional NOP/MOP partial agonists (e.g., AT-121, BU08028, and BU10038) exerted potent anti-nociception via NOP and MOP receptor activation; however, dose-limiting adverse effects associated with the MOP receptor activation, including respiratory depression, itch sensation, physical dependence, and abuse liability, were not observed. Moreover, a mixed NOP/opioid receptor agonist, cebranopadol, presented promising outcomes in clinical trials as a novel analgesic. Collectively, the dual agonistic actions on NOP and MOP receptors, with appropriate binding affinities and efficacies, may be a viable strategy to develop innovative and safe analgesics.
Following the identification of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) as an endogenous ligand for the NOP receptor, ample evidence has revealed unique functional profiles of the N/OFQ‐NOP receptor system. NOP receptors are expressed in key neural substrates involved in pain and reward modulation. In nonhuman primates (NHPs), NOP receptor activation effectively exerts antinociception and anti‐hypersensitivity at the spinal and supraspinal levels. Moreover, NOP receptor activation inhibits dopaminergic transmission and synergistically enhances mu‐opioid peptide (MOP) receptor‐mediated analgesia. In this article, we have discussed the functional profiles of ligands with dual NOP and MOP receptor agonist activities and highlight their optimal functional efficacy for pain relief and drug abuse treatment. Through coactivation of NOP and MOP receptors, bifunctional NOP/MOP receptor “partial” agonists (e.g., AT‐121, BU08028, and BU10038) reveal a wider therapeutic window with fewer side effects. These newly developed ligands potently induce antinociception without MOP receptor agonist‐associated side effects such as abuse potential, respiratory depression, itching sensation, and physical dependence. In addition, in both rodent and NHP models, bifunctional NOP/MOP receptor agonists can attenuate reward processing and/or the reinforcing effects of opioids and other abused drugs. While a mixed NOP/opioid receptor “full” agonist cebranopadol is undergoing clinical trials, bifunctional NOP/MOP “partial” agonists exhibit promising therapeutic profiles in translational NHP models for the treatment of pain and opioid abuse. This class of drugs demonstrates the therapeutic advantage of NOP and MOP receptor coactivation, indicating a greater potential for future development.
Learning objectives:
After participating in this activity, learners should be better able to:• Identify the effects of dysregulated opioid signalling in depression• Evaluate the use of opioid compounds and ketamine in patients with depression ABSTRACT: Major depressive disorder (MDD) remains one of the leading causes of disability and functional impairment worldwide. Current antidepressant therapeutics require weeks to months of treatment prior to the onset of clinical efficacy on depressed mood but remain ineffective in treating suicidal ideation and cognitive impairment. Moreover, 30%-40% of individuals fail to respond to currently available antidepressant medications. MDD is a heterogeneous disorder with an unknown etiology; novel strategies must be developed to treat MDD more effectively. Emerging evidence suggests that targeting one or more of the four opioid receptors-mu (MOR), kappa (KOR), delta (DOR), and the nociceptin/orphanin FQ receptor (NOP)-may yield effective therapeutics for stress-related psychiatric disorders. Furthermore, the effects of the rapidly acting antidepressant ketamine may involve opioid receptors. This review highlights dysregulated opioid signaling in depression, evaluates clinical trials with opioid compounds, and considers the role of opioid mechanisms in rapidly acting antidepressants.
The opioid‐like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor NOP contribute to Parkinson's disease (PD) and motor complications associated with levodopa‐therapy. The N/OFQ‐NOP receptor system is expressed in cortical and subcortical motor areas and, notably, in dopaminergic neurons of the substantia nigra compacta. Dopamine depletion as in rodent models of PD results in upregulation of N/OFQ transmission in SN and downregulation of N/OFQ transmission in striatum. Consistent with this, NOP receptor antagonists relieve motor deficits in PD models by re‐instating the physiological balance between excitatory and inhibitory inputs impinging on nigro‐thalamic GABAergic neurons. NOP receptor antagonists also counteract the degeneration of nigro‐striatal dopaminergic neurons, possibly by attenuating the excitotoxicity or modulating the immune response. Conversely, NOP receptor agonists attenuate levodopa‐induced dyskinesia by attenuating the hyperactivation of striatal D1 signalling in neurons of the direct striato‐nigral pathway. The N/OFQ‐NOP receptor system might represent a novel target in the therapy of PD.
The neuropeptide nociceptin/orphanin FQ (N/OFQ) can be released by stressors and is associated with disorders of emotion regulation and reward processing. N/OFQ and its receptor, NOP, are enriched in dopaminergic pathways, and intra-ventricular agonist delivery decreases dopamine levels in the dorsal striatum, nucleus accumbens (NAc), and ventral tegmental area (VTA). We used whole cell electrophysiology in acute rat midbrain slices to investigate synaptic actions of N/OFQ. N/OFQ was primarily inhibitory, causing outward currents in both immunocytochemically identified dopaminergic (tyrosine hydroxylase positive (TH(+)) and non-dopaminergic (TH(−)) VTA neurons (effect at 1 μM: 20 ± 4 pA). Surprisingly, this effect was mediated by augmentation of postsynaptic GABA A R currents, unlike the substantia nigra pars compacta (SNc), where the N/OFQ induced outward currents were K ⁺ channel dependent. A smaller population, 19% of all VTA neurons, responded to low concentrations N/OFQ with inward currents (10 nM: −11 ± 2 pA). Following 100 nM N/OFQ, the response to a second N/OFQ application was markedly diminished in VTA neurons (14 ± 10% of first response), but not in SNc neurons (90 ± 20% of first response). N/OFQ generated outward currents in medial prefrontal cortex (mPFC)-projecting VTA neurons, but inward currents in a subset of posterior anterior cingulate cortex-projecting VTA neurons. While N/OFQ inhibited NAc-projecting VTA cell bodies, it had little effect on electrically or optogenetically evoked terminal dopamine release in the NAc measured ex vivo with fast scan cyclic voltammetry. These results extend our understanding of the N/OFQ system in brainstem circuits implicated in many neurobehavioral disorders.
Significance statement
The neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are engaged under conditions of stress and are associated with reward processing disorders. Both peptide and receptor are highly enriched in ventral tegmental area (VTA) pathways underlying motivation and reward. Using whole cell electrophysiology in rat midbrain slices we found: 1) NOPs are functional on both dopaminergic and non-dopaminergic VTA neurons; 2) N/OFQ differentially regulates VTA neurons based on neuroanatomical projection target; and 3) repeated application of N/OFQ produces evidence of receptor desensitization in the VTA but not the SNc. These results reveal candidate mechanisms by which the NOP system regulates motivation and emotion.
Background:
Posttraumatic stress disorder (PTSD) is a stress disorder that develops in only some individuals following a traumatic event. Data suggest that a substantial fraction of women recover after sexual violence. Thus, the investigation of stress and antistress neuropeptides in this sample has the potential to inform the neurochemistry of resilience following trauma. Nociceptin is an antistress neuropeptide in the brain that promotes resilience in animal models of PTSD.
Methods:
[11C]NOP-1A positron emission tomography was used to measure the in vivo binding to nociceptin receptors in 18 college women who had experienced sexual violence irrespective of whether they met DSM-5 diagnostic criteria for PTSD. [11C]NOP-1A data from 18 healthy control subjects were also included to provide a contrast with the sexual violence group. [11C]NOP-1A total distribution volume (VT) in the regions of interest were measured with kinetic analysis using the arterial input function. The relationships between regional VT and Clinician-Administered PTSD Scale for DSM-5 total symptom and subscale severity were examined using correlational analyses.
Results:
No differences in [11C]NOP-1A VT were noted between the sexual violence and control groups. VT in the midbrain and cerebellum were positively correlated with PTSD total symptom severity in the past month before positron emission tomography. Intrusion/re-experiencing and avoidance subscale symptoms drove this relationship. Stratification of subjects by a DSM-5 PTSD diagnosis and contrasting their VT with that in control subjects showed no group differences.
Conclusions:
Decreased midbrain and cerebellum nociceptin receptors are associated with less severe PTSD symptoms. Medications that target nociceptin should be explored to prevent and treat PTSD.
Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely, Ser ³⁴⁶ , Ser ³⁵¹ , Thr ³⁶² , and Ser ³⁶³ , and proceeded with a temporal hierarchy, with Ser ³⁴⁶ as the first site of phosphorylation. G protein–coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which, in turn, facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein–dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.
Nociceptin/orphanin FQ (N/OFQ) is an endogenous neuropeptide of 17 amino acids, related to opioid peptides but with its own receptor, distinct from conventional opioid receptors, the ORL1 or NOP receptor. The NOP receptor is a G protein-coupled receptor which activates Gi/o proteins and thus induces an inhibition of neuronal activity. The peptide and its receptor are widely expressed in the central nervous system with a high density of receptors in regions involved in learning and memory. This review describes the consequences of the pharmacological manipulation of the N/OFQ system by NOP receptor ligands on learning processes and on the consolidation of various types of long-term memory. We also discuss the role of endogenous N/OFQ release in the modulation of learning and memory. Finally we propose several putative neuronal mechanisms taking place at the level of the hippocampus and amygdala and possibly underlying the behavioral amnestic or promnesic effects of NOP ligands.
Addiction is composed of three phases: intoxication, withdrawal, and craving. Negative reinforcement, strengthening a behaviour by removing an aversive stimulus, has been associated with the withdrawal phase. An imbalance of neurotransmitters within the brain’s stress (nociceptin, neuropeptide Y) and anti-stress (CRF, norepinephrine, etc.) system is attributed to negatively reinforced compulsive behaviours associated with relapse. Similarly, post-traumatic stress disorder is characterized by an overactive stress system. In a PTSD mouse model, rodents exhibited impaired cued-fear memory consolidation when nociceptin transmission was blocked. Furthermore, a single-nucleotide polymorphism has been identified between women diagnosed with PTSD and the severity of PTSD symptoms, suggesting a genetic basis. Therefore, it is critical to understand the functions and interactions between the brain’s stress and anti-stress neurotransmitters, specifically nociceptin. This paper will examine the hypothalamic-pituitary-adrenocortical axis, evaluate the functions of corticotropin-releasing-factor and nociceptin, discuss nociceptin’s role as an anxiolytic or anxiogenic, and discuss PET-imaging studies—all of which targeted nociceptin receptors (NOP-R). Finally, the discussion of pharmacological interventions will be proposed as preventative or therapeutic treatments for those suffering from PTSD and substance-use disorders.
Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) were identified in the mid 90s as a novel peptidergic system structurally related to opioids. A growing body of preclinical evidence suggests that blockade of NOP receptors evokes antidepressant-like actions. These have been explored using a range of compounds (peptide and non peptide antagonists), across different species (rat and mouse) and assays (behavioural despair and chronic mild stress) suggesting a robust and consistent antidepressant-like effect. Moreover, rats and mice knockout for the NOP receptor gene display an antidepressant-like phenotype in behavioural despair assays. Electrophysiological, immunohistochemical and neurochemical studies point to an important role played by monoaminergic systems, particularly 5-HTergic, in mediating the antidepressant-like properties of NOP antagonists. However other putative mechanisms of action, including modulation of the CRF system, circadian rhythm and a possible neuroendocrine-immune control might be involved. A close relationship between the N/OFQ-NOP receptor system and stress responses is well described in the literature. Stressful situations also alter endocrine, behavioural and neurochemical parameters in rats and chronic administration of a NOP antagonist restored these alterations. Interestingly, clinical findings showed that plasma N/OFQ levels were significantly altered in major and post-partum depression, and bipolar disease patients. Collectively, data in the literature support the notion that blockade of NOP receptor signaling could be a novel and interesting strategy for the development of innovative antidepressants.
Nociceptin and nocistatin are endogenous ligands of G protein coupled receptor family. Numerous techniques have been used to study the diverse parameters including, localization, distribution and ultrastructure of these peptides. The majority of the studies parameters are based on their physiological roles in different organ systems. The present study presents an overview of the different methods used for the study of nociceptin, nocistatin and their receptors. Nociceptin has been implicated in many physiological functions including, nociception, locomotion, stressed-induced analgesia, learning and memory, neurotransmitter and hormone release, renal function, neuronal differentiation, sexual and reproductive behavior, uterine contraction, feeding, anxiety, gastrointestinal motility, cardiovascular function, micturition, cough, hypoxic-ischemic brain injury, diuresis and sodium balance, temperature regulation, vestibular function, and mucosal transport. It has been noted that the use of light and electron microscopy was less frequent, though it may be one of the most promising tools to studying the intracellular localization of these neuropeptides. In addition, more studies on the level of circulating nociceptin and nocistatin are also necessary for investigating their clinical roles in health and disease. A variety of modern tools including physiological, light and electron microscopy (EM) are needed to decipher the extent of intracellular localization, tissue distribution and function of these peptides. The intracellular localization of nociceptin and nocistatin will require a high resolution transmission EM capable of identifying these peptides and other supporting molecules that co-localize with them. A tracing technique could also elucidate a possible migratory ability of nociceptin and nocistatin from one cellular compartment to the other.
In the present study we investigated whether the neuropeptide nociceptin/orphanin FQ (N/OFQ), previously implicated in the pathogenesis of Parkinson's disease, also affects L-DOPA-induced dyskinesia. In striatal slices of naive rodents, N/OFQ (0.1-1 mum) prevented the increase of ERK phosphorylation and the loss of depotentiation of synaptic plasticity induced by the D1 receptor agonist SKF38393 in spiny neurons. In vivo, exogenous N/OFQ (0.03-1 nmol, i.c.v.) or a synthetic N/OFQ receptor agonist given systemically (0.01-1 mg/Kg) attenuated dyskinesias expression in 6-hydroxydopamine hemilesioned rats primed with L-DOPA, without causing primary hypolocomotive effects. Conversely, N/OFQ receptor antagonists worsened dyskinesia expression. In vivo microdialysis revealed that N/OFQ prevented dyskinesias simultaneously with its neurochemical correlates such as the surge of nigral GABA and glutamate, and the reduction of thalamic GABA. Regional microinjections revealed that N/OFQ attenuated dyskinesias more potently and effectively when microinjected in striatum than substantia nigra (SN) reticulata, whereas N/OFQ receptor antagonists were ineffective in striatum but worsened dyskinesias when given in SN. Quantitative autoradiography showed an increase in N/OFQ receptor binding in striatum and a reduction in SN of both unprimed and dyskinetic 6-hydroxydopamine rats, consistent with opposite adaptive changes of N/OFQ transmission. Finally, the N/OFQ receptor synthetic agonist also reduced dyskinesia expression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated dyskinetic macaques without affecting the global parkinsonian score. We conclude that N/OFQ receptor agonists may represent a novel strategy to counteract L-DOPA-induced dyskinesias. Their action is possibly mediated by upregulated striatal N/OFQ receptors opposing the D1 receptor-mediated overactivation of the striatonigral direct pathway.
The studies proposed in this project will test the hypotheses that in the non-human primate (1) the functions and behavioral effects of the NOP receptor are independent of classical opioid receptors, (2) activation of the NOP receptor produces strong antinociception without abuse liability, and (3) NOP receptor agonists possess a promising therapeutic profile as analgesics compared to mu opioids following repeated administration in primates. Several key findings have been obtained and some have been published. First, intrathecal administration of N/OFQ only produced antinociception in primates. The functional profiles of spinal NOP receptors are different between primates and rodents. Second, intrathecal administration of N/OFQ and other NOP receptor agonists produced antinociception without eliciting itch/scratching responses, indicating that NOP receptor agonists represent a therapeutic target as spinal analgesics. Third, NOP receptor agonists produced antinociceptive effects comparable to clinically used mu opioids such as morphine and alfentanil in three different primate pain models, indicating that the analgesic effectiveness of NOP receptor agonists may be similar to that of mu opioid analgesics in humans. Finally, unlike mu opioids, NOP receptor agonists did not produce reinforcing effects, respiratory depressant, sedation, or itch/pruritic side effects, indicating that NOP receptor agonists may be a new generation of novel analgesics without abuse liability.
In 1994, the isolation of an opioid receptor-related clone soon led to the isolation and characterization of a novel neuropeptide, termed nociceptin or orphanin FQ (N/OFQ). This heptadecapeptide binds to the N/OFQ receptor (NOP) with high affinity, but does not interact directly with classical opioid receptors. The regional distribution of N/OFQ and of its receptor suggest any possible involvement of this neurotransmission system in motor and balance control, reinforcement and reward, nociception, stress response, sexual behavior, aggression and autonomic control of physiological processes as well as of immune functions. The actions of N/OFQ may also be uniquely dependent on contextual factors, both genetic and environmental. As for most of the G protein coupled receptors, NOP C-terminal sequences are believed to interact with proteins that are mandatory for anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding. Increasing details of how NOP receptors are activated and removed from the plasma membrane have been elucidated in vitro, and more importantly in a physiological context. Details of how these receptors travel and recycle following internalization have also shed light on the importance of such mechanisms for any potential therapeutic use of NOP ligands. A picture of the pathways and proteins involved in these processes is beginning to emerge. This review will address molecular events contributing to NOP receptor signaling and trafficking.
Antagonism of the central opioid receptor like-1 receptor (ORL1) has been implicated in cognition, and has been a focus of drug discovery efforts to ameliorate the cognitive deficits that remain during the stable treatment of schizophrenia with current antipsychotics. In order to facilitate dose selection for phase II clinical testing an ORL1-specific PET tracer was developed to determine drug plasma concentration versus occupancy relationships in order to ensure that the doses selected and the degree of target engagement were sufficient to ensure adequate proof of concept testing. MK-0911 is a selective, high affinity antagonist for the ORL1 receptor radiolabeled with high specific activity (18)F for positron emission tomography (PET) studies. Evaluation of [(18)F]MK-0911 in rhesus monkey PET studies showed a pattern of brain uptake which was consistent with the known distribution of ORL1. In vitro autoradiography with [(18)F]MK-0911 in rhesus monkey and human brain tissue slices showed a regional distribution that was consistent with in vivo imaging results in monkey. Pre-treatment of rhesus monkeys with high doses of structurally diverse ORL1 antagonists MK-0584, MK-0337, or MK-5757 achieved blockade of [(18)F]MK-0911 in all grey matter regions. Baseline PET studies with [(18)F]MK-0911 in healthy human subjects showed tracer distribution and kinetics similar to that observed in rhesus monkey. Quantification of [(18)F]MK-0911 uptake in repeat human baseline PET studies showed a test-retest variability in volume of distribution (V(T)) averaging 3% across brain regions. Humans dosed orally with MK-5757 showed reduced [(18)F]MK-0911 tracer concentration in brain proportional with MK-5757 dose and plasma level. [(18)F]MK-0911 was useful for determining MK-5757-induced receptor occupancy of ORL1 to guide MK-5757 dose-selection for clinical proof-of-concept studies. Additionally, [(18)F]MK-0911 may be a useful tool for studying the pharmacology of ORL1 in various human populations and disease states.
The ORL1 receptor, an orphan receptor whose human and murine complementary DNAs have recently been characterized, structurally resembles opioid receptors and is negatively coupled with adenylate cyclase. ORL1 transcripts are particularly abundant in the central nervous system. Here we report the isolation, on the basis of its ability to inhibit the cyclase in a stable recombinant CHO(ORL1+) cell line, of a neuropeptide that resembles dynorphin A9 and whose amino acid sequence is Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln. The rat-brain cDNA encodes the peptide flanked by Lys-Arg proteolytic cleavage motifs. The synthetic heptadecapeptide potently inhibits adenylate cyclase in CHO(ORL1+) cells in culture and induces hyperalgesia when administered intracerebroventricularly to mice. Taken together, these data indicate that the newly discovered heptadecapeptide is an endogenous agonist of the ORL1 receptor and that it may be endowed with pro-nociceptive properties.
By using a reverse transcription-PCR approach we have cloned a peptide precursor from mouse brain which contains the sequence of orphanin FQ/nociceptin. The mouse sequence of orphanin FQ/nociceptin is identical at the amino acid level with that isolated from rat and porcine brain. Northern analysis of the mRNA encoding the precursor reveals a single band of approx. 1 kb, with the highest levels in the brain and much lower levels in kidney and spleen. Southern analysis is consistent with a single gene. The precursor peptide from mouse contains two putative peptides. Upstream from the orphanin FQ/nociceptin is a 41-amino-acid peptide which is almost identical, except for a six-amino-acid insertion, with the corresponding 35-amino-acid peptide predicted from the rat sequence. Interestingly, the mouse contains a triple AEPGAD repeat within this peptide that is not seen in the rat sequence. Downstream from the orphanin FQ/nociceptin sequence is another 17-amino-acid peptide which is identical with that found in the rat.
The heptadecapeptide orphanin FQ (OFQ) is a recently discovered neuropeptide that exhibits structural features reminiscent of the opioid peptides and that is an endogenous ligand to a G protein-coupled receptor sequentially related to the opioid receptors. We have cloned both the human and rat cDNAs encoding the OFQ precursor proteins, to investigate whether the sequence relationships existing between the opioid and OFQ systems are also found at the polypeptide precursor level, in particular whether the OFQ precursor would encode several bioactive peptides as do the opioid precursors, and to study the regional distribution of OFQ sites of synthesis. The entire precursor protein displays structural homology to the opioid peptide precursors, especially preprodynorphin and preproenkephalin. The predicted amino acid sequence of the OFQ precursor contains a putative signal peptide and one copy of the OFQ sequence flanked by pairs of basic amino acid residues. Carboxyl-terminal to the OFQ sequence, the human and rat precursors contain a stretch of 28 amino acids that is 100% conserved and thus may encode novel bioactive peptides. Two peptides derived from this stretch were synthesized but were found to be unable to activate the OFQ receptor, suggesting that if they are produced in vivo, these peptides would likely recognize receptors different from the OFQ receptor. To begin analyzing the sites of OFQ mRNA synthesis, Northern analysis of human and rat tissues were carried out and showed that the OFQ precursor mRNA is mainly expressed in the brain. In situ hybridization of rat brain slices demonstrated a regional distribution pattern of the OFQ precursor mRNA, which is distinct from that of the opioid peptide precursors. These data confirm that the OFQ system differs from the opioid system at the molecular level, although the OFQ and opioid precursors may have arisen from a common ancestral gene.
Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.
The putative role of the opioid system in cognitive and memory functions prompted us to search for possible changes in the cohort of the major opioid receptors, μ, δ and κ, in Alzheimer’s disease. The present study examines alterations in opioid receptor levels by quantitative autoradiography. These experiments were carried out on coronal sections of postmortem brains from Alzheimer’s disease patients and from aged-matched, dementia-free individuals. Brain sections were labeled with the tritiated forms of μ-, δ- and κ-opioid ligands; DAMGO ([d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin), DPDPE ([d-Pen2,5]-enkephalin) and bremazocine (in the presence of μ- and δ-ligands), respectively. Nonspecific binding was determined in the presence of naloxone (10 μM). Brain areas analyzed were caudate, putamen, amygdaloid complex, hippocampal formation and various cerebral and cerebellar cortices. Image analyses of autoradiographs show, that in comparison to the same areas in control brain, statistically significant reductions in μ-opioid receptor binding occur in the subiculum and hippocampus of Alzheimer’s disease brains. Binding of δ-opioid receptors is also decreased in the amygdaloid complex and ventral putamen of Alzheimer’s disease brains. In contrast, large increases of κ-opioid receptor binding are found in the dorsal and ventral putamen as well as in the cerebellar cortex of Alzheimer’s disease brains. Levels of μ-, δ- and κ-opioid receptor binding are unaltered in the caudate, parahippocampal gyrus and occipito–temporal gyrus. These results may suggest an involvement of the endogenous opioid system in some of the multitude of effects that accompany this dementia.
The cloning of the delta-opioid receptor allowed for the rapid cloning of the two other classically defined opioid receptors, the mu- and kappa-opioid receptors. However, several groups cloned a fourth receptor (ORL-1, for opioid receptor-like) that had high homology to the opioid receptors but did not bind any known endogenous opioid peptides (i.e., endorphins) or exogenous opiates. Recently, two independent groups isolated a 17-amino-acid peptide that is an endogenous ligand for ORL-1; one group named it orphanin FQ (OFQ), the other named it nociceptin (N). It was reported that intracerebroventricular administration of this heptadecapeptide (OFQ/N) in mice induced an increased responsiveness to painful stimuli, an effect in striking contrast to the analgesia that is a hallmark of classical opiate drugs. Further research has revealed that OFQ/N has complex effects on pain perception: OFQ/N has been touted as having analgesic, hyperalgesic, and antiopioid properties. In addition to discussing these disparate findings, this review highlights the structural and pharmacological parallels between ORL-1 and opioid receptors as well as their respective endogenous ligands.
The endogenous ligand for the orphan opioid receptor, orphanin FQ/nociceptin (OFQ), has recently been characterized. The OFQ peptide sequence contains paired basic amino acids, suggesting the possibility of posttranslational processing to a peptide containing the first 11 amino acids of the OFQ peptide. This peptide has been reported in the brain and it has a unique pharmacology. In the present study, we compared the autoradiographic distribution of 125I[Tyr14]OFQ and 125I[Tyr10]OFQ(1–11) in coronal rat brain sections. Nonspecific binding was defined with unlabeled OFQ or OFQ(1–11), respectively. Both radioligands demonstrated high levels of specific binding (>95% of total binding), with no appreciable binding in white matter areas with either ligand. 125I[Tyr14]OFQ binding was widely distributed throughout the rat brain. In contrast, 125I[Tyr10]OFQ(1–11) binding was more restricted. The highest 125I[Tyr14]OFQ binding levels measured in this study were found in the locus coeruleus, an area which contained very low 125I[Tyr10]OFQ(1–11) binding. Both ligands labeled the cortex, hippocampus and amygdala. In the thalamus, 125I[Tyr14]OFQ binding was prominent in most nuclei, whereas 125I[Tyr10]OFQ(1–11) binding was restricted to the midline thalamus. 125I[Tyr14]OFQ binding was heavy in the suprachiasmatic hypothalamus, and moderate in other hypothalamic nuclei. 125I[Tyr10]OFQ(1–11) binding in the hypothalamus, however, was present mainly in the ventromedial hypothalamic nucleus. Lower binding levels of both ligands were found in the caudate putamen. The distinct autoradiographic patterns of these two ligands are consistent with different binding sites, which might help explain their different functional activities. J. Comp. Neurol. 423:319–329, 2000. © 2000 Wiley-Liss, Inc.
Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ-system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle-sized neurons. These neurons were scattered between large projection-type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection-type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development. J. Comp. Neurol. 399:139–151, 1998. © 1998 Wiley-Liss, Inc.
Homology cloning and, more recently, the sequencing of whole genomes, have identified many open reading frames encoding proteins of unknown function, in particular putative G protein-coupled membrane receptors. Identification of orphan receptors in this way has marked the advent of 'reverse pharmacology' to identify the corresponding physiological ligands. This approach has led to the discovery of the ORL1 (Opioid Receptor-Like 1) receptor, and of its natural ligand, nociceptin/orphanin FQ (noc/oFQ), the basic components of a new peptide-based signalling pathway in the nervous system. Based on genetic criteria, the ORL1 and opioid receptors belong to the same family, as do noc/oFQ and opioid peptides. The marked structural analogy between the ORLI and opioid receptors, especially the kappa-opioid receptor, and the noc/oFQ and opioid peptides, particularly dynorphin A, is not reflected anatomically since noc/oFQ and opioid peptides appear to be located in separate neuronal circuits. Noc/oFQ triggers the same G protein-mediated signalling pathways as do opioids, however, to produce pharmacological effects that sometimes differ from, and even oppose, those of opioids. Noc/oFQ stimulates an outward K+ current and/or inhibits voltage-gated Ca2+ channels, thereby reducing synaptic efficacy, i.e. neuronal activity. In the rat, noc/oFQ is endowed with supraspinal pronociceptive/anti-opioid properties (it suppresses opioid-mediated analgesia), while convergent electrophysiological and behavioural data indicate that the peptide is a spinal analgesic. Noc/oFQ has not yet been found to precipitate withdrawal in morphine-tolerant rats. Nor does it elicit motivational effects, suggesting it lacks abuse liability. Also, by acting supraspinally, noc/oFQ impairs motor performance, suppresses spatial learning, induces feeding, and regulates basal and stress-induced release of pituitary hormones. Noc/oFQ is also active when administered intravenously, exhibiting potent smooth muscle relaxant, diuretic, and antinatriuretic properties. Last but not least, noc/oFQ appears to regulate stimulated immune function, and to be involved in neuronal differentiation. The discovery of noc/oFQ, a neuropeptide with multiple functions, will certainly improve our knowledge of brain physiology, and may find therapeutic applications, for example in the management of pain or hyponatremic and water-retaining diseases. However, given the wide distribution of noc/oFQ and its receptor, the pharmacological profile of noc/oFQ is likely to be incomplete, and other as yet unknown functions of the peptide remain to be discovered. Most helpful in this respect will be the identification of new ligands of the ORL1 receptor, particularly antagonists. If research on noc/oFQ carries on unabated at the present pace, potentially clinically interesting new compounds could become available in the not too distant future.
We have examined the distribution of the opioid receptor-like-1 (ORL-1) transcript in the human CNS as well as human immune cells by RT-PCR and RNAse protection. The hORL-1 mRNA was distributed throughout the brain and particularly abundant in cortical areas, striatum, thalamus and hypothalamus. In the immune system, gene transcription was observed in normal circulating lymphocytes and monocytes as well as in T, B and monocytic cell lines. A splice variant, lacking 15 nucleotides at the junction between exon 1 and exon 2, showed a distribution similar to the already known ORL-1 transcript. Altogether these results show comparable expression levels of the hORL-1 gene in both nervous and immune systems, suggesting that the ORL-1-encoded receptor may participate to neuronal and non-neuronal physiological functions in humans.
A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP- binding
protein (G protein)- coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ,
has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated
adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid
ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high
affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not
induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin
FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.
The cloning of the opioid receptors has profoundly affected our understanding of opioid-receptor expression, regulation and function. This review focuses on the impact that cloning has had on our understanding of opioid-receptor anatomy, and provides broad anatomical maps of the three opioid-receptor mRNAs in relation to their binding sites. In addition, three model anatomical systems, the nigrostriatal and mesolimbic dopamine systems, the hypothalamic neuroendocrine axes, and the ascending and descending pain pathways, have been highlighted to discuss issues of receptor transport, trafficking and pre- versus postsynaptic localization.
Pharmacological evidence indicates that lymphocytes express opioid receptors, but this finding has been questioned. By DNA sequencing of reverse transcription-polymerase chain reaction products, we have found that mouse lymphocytes express mRNA encoding an orphan opioid receptor. These mRNA transcripts were detected in the CD4+, CD8+, and CD4- CD8- lymphocyte subpopulations. Northern blot analysis confirmed that splenic lymphocytes express a 1.5-kb orphan opioid receptor mRNA. Fifteen bases encoding Tyr71-Arg75 in the first intracellular loop are alternatively spliced, suggesting that orphan opioid receptor mRNA encodes two receptor subtypes. Treatment of lipopolysaccharide-stimulated lymphocytes with orphan opioid receptor antisense oligonucleotides suppressed polyclonal IgG and IgM production by 50%. Our results provide direct evidence that lymphocytes express an opioid-like receptor gene, and suggest that this receptor plays a functional role in immunocompetence.
A rat brain cDNA library was screened for clones homologous to the recently cloned mouse delta-opioid receptor (DOR-1). Among the clones isolated was Hyp 8-1, a clone with a unique nucleotide sequence capable of encoding a putative protein which is 57-58% identical to the amino acid sequences of the cloned delta, mu and kappa opioid receptors, indicating a close relationship of Hyp 8-1 with the opioid receptor family. Several cDNAs representing possible splice variants of Hyp 8-1 were also isolated. Binding studies of COS-7 cells transfected with clone Hyp 8-1 failed to demonstrate specific binding with several 3H-opioid ligands. In situ hybridization studies indicate that the mRNA for Hyp 8-1 is distributed discretely throughout the rat brain, in an overall pattern which is different from that of several other G-protein-coupled seven transmembrane receptors. Thus, it is likely that the Hyp 8-1 cDNA encodes a novel peptide receptor.
Radioligand binding and cDNA homology studies have suggested the existence of opiate receptors distinct from the recently-cloned mu, delta and kappa receptors. XOR1S, a rat brain cDNA whose predicted translation product displays 67-72% homology with those encoded by mu 1, delta 1 and kappa 1 opiate receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XOR1L variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XOR1 mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of opiate ligands nor reproducible opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the opiate receptor gene family, including apparent differential splicing and expression in peripheral tissues.
Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the delta-opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane-spanning domains of a G protein-coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORL1 is 49-50% identical to the murine mu-, delta- and kappa-opioid receptors and, in CHO-K1 cells stably transfected with a pRc/CMV:hORL1 construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a 'universal' (nonselective) opiate agonist. Yet, hORL1 appears not to be a typical opioid receptor. Neither is it a somatostatin or sigma (N-allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula, ...) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.
Quantitative autoradiographic assessment of cerebral cortical laminar distribution of mu, delta and kappa opioid receptors was carried out in coronal sections of five post-mortem human brains obtained at autopsy. The cortical areas studied were: cingulate, frontal, insular, parietal, parahippocampal, temporal, occipitotemporal, occipital and striate area. In general, the laminar patterns of distribution for the three types of receptors are distinctive. Peak levels of delta opioid binding are in laminae I, II, and IIIa. mu-Receptors are located in lamina III followed by I and II in cingulate, frontal, insular and parietal cortices and lamina IV in temporal and occiptotemporal cortices. kappa-Receptors are found concentrated in laminae V and VI. The patterns of opioid binding in cortical laminae showed remarkable consistency in all five brains examined. In contrast to other cortical areas, the parahippocampal gyrus, at the level of the amygdaloid formation, demonstrated peak kappa receptor density in laminae I, II and III. mu-Opioid binding was undetectable in the lateral occipital cortex and in the striate area.
A recently identified novel peptide, orphanin FQ/nociceptin (OFQ/N), is an endogenous ligand for a unique member of the cloned opioid receptor family. Saturation studies in mouse brain membranes reveal curvilinear Scatchard plots with both a higher (KD 3.8 pM, Bmax 31.6 fmol/mg protein) and a lower (KD 896 pM, Bmax 233 fmol/mg protein) affinity site in brain tissue, compared to only a single site in transfected CHO cells (KD 36 pM). Competition studies confirm the high affinity of OFQ/N for this site, but shallow Hill slopes suggest heterogeneity. Traditional opioids have poor affinity for this receptor and OFQ/N and its fragments do not label traditional opioid receptors. In brain homogenates, both OFQ/N and OFQ/N(1-11) inhibit forskolin-stimulated camp accumulation with IC50 values of 1 nM or less, an action which is readily reversed by opioid antagonists. OFQ/N(1-7) shows little activity. Together, these studies suggest the presence of heterogeneous, functionally active OFQ/N receptors in mouse brain.
Antisera were generated against nociceptin/orphanin FQ, the putative ligand of the opioid receptor-like ORL1 receptor. Dot blot analysis showed that the antibodies selectively detect nociceptin but not dynorphin or other opioid peptides. Immunofluorescent staining of tissue sections revealed dense plexus of nociceptin-immunoreactive nerve fibres and terminals within the spinal cord dorsal horn, sensory trigeminal complex, raphe nuclei, locus coeruleus, periaqueductal grey, amygdala, habenula, hypothalamic region and septal area in mice and rats. When adjacent sections were stained either with the nociceptin antibody or the pan-opioid 3-E7 mouse monoclonal antibody, an overlapping distribution was observed in many nociceptive centres including the superficial dorsal horn, sensory trigeminal complex and periaqueductal grey. However, confocal microscopic examination of dual-labelled spinal cord and brain stem sections showed no instances of co-localization of nociceptin and opioid peptides in these regions. Intracerebroventricular administration of nociceptin has been shown to induce hyperalgesia. Thus, the present results suggest that nociceptin and opioids are released from different terminals thereby modulating pain signals in opposite ways.
The binding sites of [3H]nociceptin (also named Orphanin FQ), the endogenous ligand of the ORL1 (opiate receptor like 1) receptor, were localized in the central nervous system of the mouse using an autoradiographic procedure. A high density of binding sites was seen in the cerebral cortex, paraventricular nucleus of the thalamus, amygdaloid complex, suprachiasmatic nucleus, medial thalamus and medial geniculate nucleus. Moderate binding was observed in the nucleus accumbens, lateral septum, lateral thalamus, hippocampus, periaqueductal grey matter and pons. Finally, low levels of binding were seen in the striatum, olfactory tubercle, hypothalamus and substantia nigra. Thus, it appears that the ORL1 receptor is particularly abundant in the cerebral cortex and limbic system of the mouse brain.
[125I][Tyr14]Orphanin binds to a number of saturable non-interacting binding sites in rat brain cortical membrane preparations, with a density of 510 fmol/mg protein and affinity 0.9 nM. This high affinity, saturable [125I][Tyr14]orphanin binding was not inhibited by leu-enkephalin and by other ligands for opiate and neurotransmitter receptors both in membrane preparations and brain sections. In rat brain sections, the highest density of binding was found in the outer and medial cortical layers, subiculum, hippocampus and nucleus accumbens; intermediate binding densities were found in the inner cortical layer, pontine nuclei, thalamus and hypothalamus. Very low specific binding was seen in the cerebellum and striatum, according to the described distribution of ORL1 transcripts. These results suggest that [125I][Tyr14] orphanin binding in rat brain occurs to the described ORL1 receptor.
High affinity and saturable nociceptin (orphanin FQ) receptors were detected and characterized in adult rat and human fetal hypothalamic membranes, utilizing [125I]Tyr12-nociceptin as ligand. Nociceptin bound with picomolar affinity, dynorphin A with nanomolar affinity, naloxone and dynorphan A(1-8) with micromolar while des-Tyr1-dynorphin (dynorphin A(2-17)), several other opioids, morphine and benzomorphans failed to compete for binding at 1-10 microM. Gpp(NH)p together with sodium ion markedly decreased binding, consistent with involvement of a G protein-linked receptor.
Following the elucidation of the amino acid sequences of the mu-, delta- and kappa-opioid receptors, a new 'orphan opioid receptor' was cloned with a high degree of homology to the 'classical' opioid receptors. The endogenous opioid peptides show little or no activity at this new receptor; however, a novel endogenous peptide for the orphan opioid receptor has been isolated and sequenced. Here, Graeme Henderson and Sandy McKnight review recent findings on this new receptor and its endogenous ligand, and address the contentious issue of whether activation of this receptor results in hyperalgesia or analgesia.
The publication of the delta opioid receptor sequence led to the cloning of three homologous receptors: the mu and kappa opioid receptors, and a novel opioid-like orphan receptor. The orphan receptor's endogenous ligand, a 17-amino-acid peptide that resembles dynorphin, was named 'orphanin FQ' and 'nociceptin' (OFQ/N1-17). The OFQ/N1-17 receptor is expressed widely in the nervous system, and it is becoming clear that the peptide is likely to participate in a broad range of physiological and behavioral functions. At the cellular level, OFQ/N1-17 has much in common with the classical opioids; however, functional studies are now revealing distinct actions of this peptide. Identified only two years ago, OFQ/N1-17 has already attracted a great deal of attention. The number and diversity of papers focused on OFQ/N1-17 at the recent meeting of the Society for Neuroscience augur an exciting future for this new peptide.
Nociceptin/orphanin FQ (N/OFQ) and its receptor share similarities to opioids and their receptors in terms of the molecular structure and signaling pathway, but the two systems exhibit different actions in vivo. To understand the mechanism of N/OFQ-system actions, we examined, by in situ hybridization analysis, the distribution of preproN/OFQ and N/OFQ receptor mRNAs in the developing and adult mouse central nervous systems (CNS). In most neural regions, preproN/OFQ mRNA was mainly expressed in a small population of middle-sized neurons. These neurons were scattered between large projection-type neurons or within the neuropil, suggestive of interneurons. In some other nuclei (lateral septum, bed nucleus of the stria terminalis, reticular thalamic nucleus, inferior colliculus, and rostral periolivery nucleus), preproN/OFQ mRNA was expressed in a number of large projection-type neurons. By contrast, N/OFQ receptor mRNA was evenly expressed in most neurons of the adult CNS. Considering the inhibitory actions of N/OFQ, the distinct cellular expression pattern of the N/OFQ system suggests that the release of N/OFQ from interneurons may lower neuronal and synaptic activities of neighboring neurons, leading to integration or modulation of local circuits. Furthermore, the cellular expression pattern, distinct from that of the opioid system, may provide a possible molecular/cellular basis for the different in vivo actions of N/OFQ and opioids. In embryonic stages, both preproN/OFQ and N/OFQ receptor mRNAs were highly and widely expressed in the mantle zone, suggesting the possible importance of N/OFQ signaling in CNS development.
The presence of pairs of basic amino acids within the sequence of orphanin FQ/nociceptin (OFQ/N) peptide, the endogenous ligand for the ORL1/KOR-3 receptor, has raised the possibility that processing might generate pharmacologically important truncated peptides, including OFQ/N(1-11). OFQ/N(1-11) is pharmacologically active in vivo with a potency comparable to OFQ/N. Several tyrosine-containing analogs of OFQ/N(1-11) have been synthesized and examined for antinociceptive activity. Like OFQ/N(1-11), [Tyr1]OFQ/N(1-11), [Tyr10]OFQ/N(1-11) and [IodoTyr10]OFQ/N(1-11) given supraspinally in mice were antinociceptive in the tailflick assay in mice. The tyrosine analogs showed similar potencies as OFQ/N(1-11) but longer durations of action. This response was readily reversed by the opioid antagonist naloxone despite poor affinities for these analogs at opioid receptors. Another compound, [Tyr11]OFQ/N(1-11) was highly epileptogenic, inducing naloxone-sensitive seizures in greater than 50% of the mice tested at doses comparable to those examined with the other analogs. These results indicate that it is possible to make analgesic OFQ/N(1-11) analogs. The activity of [IodoTyr10]OFQ/N(1-11) suggests that it may prove useful as a radioligand in exploring potential OFQ/N(1-11) binding sites.
The search for novel neurotransmitters and neuropeptides has been recently revolutionized by the development of a purification strategy based on orphan G protein-coupled receptors, cloned receptors for which no natural ligands are known. This strategy uses the orphan receptor as bait to identify its natural ligand. This article will review the discovery of the first natural ligand isolated following this strategy. This ligand is a peptide that shares some striking sequence similarity to the opioid peptides and has been named Orphanin FQ or Nociceptin (OFQ/NOC). The discovery of OFQ/NOC will be described as one example of the use of orphan receptors in identifying novel neurotransmitters and neuropeptides, an example that has already been followed in the identification of other novel neuropeptides. After reviewing the conceptual and technological basis of the strategy and its successful first application, we discuss the criteria used to validate OFQ/NOC as the natural ligand of the orphan receptor and as a genuine neuropeptide. We also discuss the importance and implications of discovering OFQ/NOC mode of synthesis, which is synthesized as expected in the form of a larger polypeptide precursor, which in turn raises the question of the existence of other OFQ/NOC-related peptides. We then present an overview of the numerous studies that have blossomed after the OFQ/NOC discovery and describe the numerous physiological roles that have already been attributed to OFQ/NOC, and in particular the controversy regarding its involvement in pain perception. Because of the similarities between the OFQ/NOC and opioid systems, we also discuss overlaps between these systems and present evidence favoring a pharmacological separation between these systems. We finish by outlining the power of the orphan receptor strategy and by discussing some of its pitfalls.
Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor-like receptor (ORL-1). It and its precursor, prepro-OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro-OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes.
Little is known regarding opioid receptors in the human cerebellum. The present [11C]diprenorphine PET study investigated opioid receptor binding in the human cerebellum in vivo, and showed a differential binding level in cerebellar cortex, vermis and dentate nuclei. The additional study in vitro of opioid receptors in human cerebellar cortex and rat brain corroborated the presence of opioidergic mechanisms in the human cerebellum in contrast to the rat. A differential cellular distribution pattern was detected for the three major opioid receptors investigated. For the mu-receptor, and at a lower level for the kappa-receptor, mRNA expression was mainly observed over granule cells. Binding sites were most prominent in the molecular layer. For the delta-receptor no signal was detected. The consideration of cerebellar opioidergic mechanisms and the distribution patterns of the various opioid receptors may promote the understanding of cerebellar function and of opioidergic pharmacology in the human.
Recently, opioid receptor like1 (ORL1) receptor was identified. The ORL1 receptor is a G protein coupled receptor and the sequence of the ORL1 receptor is closely related to that of the opioid receptors. Nociceptin/orphanin FQ has been identified as a potent endogenous agonist of the ORL1 receptor and the sequence of nociceptin/orphanin FQ is closely related to that of dynorphin A. Nociceptin/orphanin FQis not active at the classical opioid receptors, such as mu, kappa and delta receptors. The distribution of prepronociceptin mRNA is distinct from that of the opioid peptide precursor. Mice lacking the ORL1 receptor showed no significant differences in nociceptive threshold compared with wild mice. The role of nociceptin/orphanin FQ on nociceptive transmission is unclear. Intracerebroventricular (i.c.v.) injection of nociceptin/orphanin FQ produced hyperalgesia and allodynia and antagonized morphine analgesia. On the other hand, intrathecal injection of low dose nociceptin/orphanin FQ produces allodynia, but high dose of nociceptin/orphanin FQ produces an analgesic effect. Although we do not fully understand the mechanisms that produce the difference between the effect of i.c.v. injection of nociceptin/orphanin FQ and that of intrathecal injection of nociceptin/orphanin FQ, we believe that spinal ORL1 receptor may be the next receptor which should be targeted by drugs designed for the treatment of pain.
The distribution of prepronociceptin messenger RNA, the recently identified endogenous ligand of the ORL1 receptor (opioid receptor-like-1), has been studied in the adult mouse central nervous system using in situ hybridization. Prepronociceptin is a new peptide precursor that generates, upon maturation, at least three bioactive peptides: nociceptin, noc2 and the recently described nocistatin. Considering both the density of labeled neurons per region and their intensity of labeling, the distribution of prepronociceptin messenger RNA-containing neurons can be summarized as follows: the highest level of prepronociceptin messenger RNA expression was detected in the septohippocampal nucleus, bed nucleus of the stria terminalis, central amygdaloid nucleus, and in selective thalamic nuclei such as the parafascicular, reticular, ventral lateral geniculate and zona incerta. High to moderate levels of prepronociceptin messenger RNA expression were detected in the lateral, ventral and medial septum, and were evident in brainstem structures implicated in descending antinociceptive pathways (e.g., the gigantocellular nucleus, raphe magnus nucleus, periaqueductal gray matter), and also observed in association with auditory relay nuclei such as the inferior colliculi, lateral lemniscus nucleus, medioventral preolivary nucleus and lateral superior nucleus. A moderate level of prepronociceptin messenger RNA expression was observed in the medial preoptic nucleus, ventromedial preoptic nucleus, periventricular nucleus, pedonculopontine tegmental nucleus, solitary tract nucleus and spinal trigeminal nucleus. A weak level of prepronociceptin messenger RNA expression was present in some areas, such as the cerebral cortex, endopiriform cortex, hippocampal formation, medial amygdaloid nucleus, anterior hypothalamic area, medial mammillary hypothalamic nuclei, retrorubral field and substantia nigra pars compacta. No labeled cells could be found in the caudate-putamen, nucleus accumbens and ventral tegmental area. The present data confirm that nociceptin is expressed in a broad array of regions of the central nervous system. In good correlation with the presently known physiological actions of nociceptin, they include, amongst others, brain areas conveying/integrating pain and auditory sensory afferences.
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.
The presence of pairs of basic amino acids within the orphanin FQ/Nociceptin (OFQ/N) sequence has raised the possibility that truncated versions of the peptide might be physiologically important. OFQ/N(1-11) is pharmacologically active in mice, despite its poor affinity in binding assays (K(i) > 250 nM) for the OFQ/N receptor. Using an analog of OFQ/N(1-11), [(125)I][Tyr(10)]OFQ/N(1-11), we identified a high-affinity binding site (K(D) 234 pM; B(max) 43 fmol/mg protein) with a selectivity profile distinct from the OFQ/N receptor and all the traditional opioid receptors. This site had very high affinity for OFQ/N and its related peptides. The most striking differences between the new site and the OFQ/N receptor previously observed in brain were seen with traditional opioids. Dynorphin A analogs and alpha-neoendorphin competed with [(125)I][Tyr(10)]OFQ/N(1-11) binding in mouse brain with K(i) values below 10 nM, while naloxone benzoylhydrazone (K(i) 3.9 nM) labeled the [(125)I][Tyr(10)]OFQ/N(1-11) binding site as potently as many traditional opioid receptors. Several other opioids, including fentanyl, (-)cyclazocine, levallorphan, naltrindole, and diprenorphine, also displayed moderate affinities for this site. Finally, the [(125)I][Tyr(10)]OFQ/N(1-11) site had a unique regional distribution consistent with a distinct receptor. Thus, [(125)I][Tyr(10)]OFQ/N(1-11) labels a novel site in brain with a selectivity profile intermediate between that of either opioid or OFQ/N receptors.
The recently discovered peptide nociceptin/orphanin FQ (N/OFQ) and its receptor NOR share many structural similarities with the opioid peptides and their receptors. The anatomical distributions of N/OFQ and NOR are similar to those of opioid peptides and receptors. In addition, NOR and opiate receptors couple via the same G-proteins to similar effectors, such as Ca(2+) channels, K(+) channels, adenylyl cyclase, and several protein kinases. Thus, the behavioral effects of N/OFQ have been investigated in the context of known opiate effects, and a possible connection has been sought between the effects of these two homologous signaling systems. Originally characterized as a nociception-producing peptide, N/OFQ has now been shown to have diverse effects on nociception, as well as effects on many other behaviors. With regard to nociception, the peptide has been reported to produce hyperalgesia, reversal of opioid-mediated analgesia, analgesia, and allodynia. N/OFQ also has effects on other behaviors, such as locomotion, feeding, anxiety, spatial attention, reproductive behaviors, and opiate tolerance. The relationship between opiates and N/OFQ is strengthened by the fact that opiates also affect these behaviors. However, the exact nature of the relationship of N/OFQ with opiates-opiate-like versus antiopiate-remains controversial. This review will detail the diverse effects of N/OFQ and suggest that this peptide, like other putative antiopiate peptides, can be described as 'opiate modulating. '
Opioid receptor-like protein ORL1, the receptor for the neuropeptide nociceptin (also named orphanin FQ), has two alternatively spliced isoforms in the rat. This alternative splicing event is generated by retaining of intron 3, 81 bases in length, in the mRNA region encoding the second extracellular loop of ORL1. A full-length rat ORL1 receptor has 367 amino acid residues. However, as revealed by sequencing of rat ORL1 genomic DNA and cDNA, the insertion of the unspliced intron 3 brings in an in-frame stop codon and, therefore, creates a truncated open-reading frame encoding only the N-terminal half of ORL1 (from the N-terminus to an alternate extracellular tail C-terminal to the fourth transmembrane domain). The two alternatively spliced transcripts are differentially expressed in tissues. In transfected mammalian cells, the full-length ORL1 displays high-affinity and selective binding for nociceptin, and inhibits the production of cyclic AMP. In contrast, the truncated ORL1 binds nociceptin and other opioid peptides very poorly and non-selectively (affinity in micromolar range), and it does not mediate any inhibitory effects on cyclic AMP production. Apparently, this truncated ORL1 does not function as a receptor for nociceptin or other ligands tested. Such alternative splicing to create a truncated ORL1 receptor might be an endogenous mechanism to negatively regulate nociceptin/ORL1 functions.
Nociceptin/orphanin FQ (N/OFQ) is an opioid-like heptadecapeptide agonist for the opioid receptor homolog, N/OFQ receptor. To explore the precise distribution of the peptide-receptor system, the authors examined the brain and spinal cord from receptor-deficient mice bearing the targeted mutation (morc(m1)), a lacZ insertional mutation in the N/OFQ receptor gene. Precursor protein N/OFQ (preproN/OFQ) mRNA was detected by using in situ hybridization, and the N/OFQ receptor was detected by using X-gal histochemistry. The N/OFQ receptor reflected by lacZ expression was observed at high levels in the dentate gyrus, lateral septum, subparafascicular thalamic nucleus, medial preoptic area, median preoptic nucleus, ventromedial preoptic nucleus, anterior hypothalamic area, paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, auditory brainstem nuclei, pontine dorsal tegmentum, and nucleus of the solitary tract. In situ detection of the N/OFQ receptor mRNA by digoxigenin-labeled riboprobes coupled with tyramide signal amplification in normal and wild-type mice resulted in the regional distribution paralleling the lacZ expression in these regions. PreproN/OFQ mRNA was expressed at high levels in the subparafascicular thalamic nucleus, central gray, central tegmental field, auditory brainstem nuclei, caudal spinal trigeminal nucleus, and spinal dorsal horn. Furthermore, variable levels of expression of the peptide and receptor were seen in distinct sites of the brain and spinal cord. These data indicate a correspondence of the peptide and the receptor in local distribution at limbic, hypothalamic, and brainstem sites. Together with concurrent physiologic and behavioral studies in mutant mice, the results suggest functional roles for the N/OFQ system, including the central regulation of learning and memory, hearing ability, water balance, food intake, and blood pressure.
A review of the binding studies performed on the receptor (ORL) for Orphanin FQ/Nociceptin is presented. Binding studies have been conducted using a variety of receptor sources: cell lines expressing the cloned receptor, cell lines endogenously expressing the receptor, and brain and other tissue from several different species. Binding studies of opioids, new ligands and antagonists at the ORL receptor are briefly discussed. Saturation, competition and binding kinetic experiments, and the effects of buffer composition are reviewed. There are numerous instances of conflicting data in published reports on OFQ; the basis for these disparities is as yet undetermined. This review endeavors to compile the results and conditions employed in binding studies as an aid to current and new researchers in this field. In an attempt to explain binding disparities, we have determined that Orphanin/Nociceptin binds to glass fiber filtermats in a "specific" manner; these new data are presented.
The ORL1 receptor is a G protein-coupled receptor structurally related to the opioid receptors, whose endogenous ligand is the heptadecapeptide nociceptin/orphanin FQ. In this review, data which have contributed to the mapping of the anatomic distribution of the ORL1 receptor have been collated with an emphasis on their relation to physiological functions. The ORL1 receptor is widely expressed in the central nervous system, in particular in the forebrain (cortical areas, olfactory regions, limbic structures, thalamus), throughout the brainstem (central periaqueductal gray, substantia nigra, several sensory and motor nuclei), and in both the dorsal and ventral horns of the spinal cord. Regions almost devoid of ORL1 receptors are the caudate-putamen and the cerebellum. ORL1 mRNA and binding sites exhibit approximately the same distribution pattern, indicating that the ORL1 receptor is located on local neuronal circuits. The ORL1 receptor is also expressed at the periphery in smooth muscles, peripheral ganglia, and the immune system. The anatomic distribution of ORL1 receptor suggests a broad spectrum of action for the nociceptin/orphanin FQ system (sensory perception, memory process, emotional behavior, etc.).
Nociceptin/orphanin FQ and nocistatin are two neuropeptides with opposing effects on spinal neurotransmission and nociception. Nociceptin/orphanin FQ selectively suppresses excitatory glutamatergic neurotransmission, while nocistatin selectively interferes with glycinergic and gamma-aminobutyric acid (GABA)-ergic transmission. Here, we performed whole-cell patch-clamp recordings from superficial rat spinal cord dorsal horn neurons to investigate the role of the opioid receptor-like (ORL)1 receptor for modulatory actions of these peptides. The partial ORL1 receptor antagonist [phe1psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)NH(2) competitively reversed the effects of nociceptin/orphanin FQ on excitatory neurotransmission (estimated pA(2) 6.43), but left the suppression of inhibitory synaptic transmission by nocistatin unaffected. These results indicate that the inhibitory action of nociceptin/orphanin FQ on glutamatergic transmission is mediated via ORL1 receptors, while nocistatin acts via a different so far unidentified receptor.
The putative role of the opioid system in cognitive and memory functions prompted us to search for possible changes in the cohort of the major opioid receptors, mu, delta and kappa, in Alzheimer's disease. The present study examines alterations in opioid receptor levels by quantitative autoradiography. These experiments were carried out on coronal sections of postmortem brains from Alzheimer's disease patients and from aged-matched, dementia-free individuals. Brain sections were labeled with the tritiated forms of mu-, delta- and kappa-opioid ligands; DAMGO ([D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin), DPDPE ([D-Pen2,5]-enkephalin) and bremazocine (in the presence of mu- and delta-ligands), respectively. Nonspecific binding was determined in the presence of naloxone (10 microM). Brain areas analyzed were caudate, putamen, amygdaloid complex, hippocampal formation and various cerebral and cerebellar cortices. Image analyses of autoradiographs show, that in comparison to the same areas in control brain, statistically significant reductions in mu-opioid receptor binding occur in the subiculum and hippocampus of Alzheimer's disease brains. Binding of delta-opioid receptors is also decreased in the amygdaloid complex and ventral putamen of Alzheimer's disease brains. In contrast, large increases of kappa-opioid receptor binding are found in the dorsal and ventral putamen as well as in the cerebellar cortex of Alzheimer's disease brains. Levels of mu- delta- and kappa-opioid receptor binding are unaltered in the caudate, parahippocampal gyrus and occipito-temporal gyrus. These results may suggest an involvement of the endogenous opioid system in some of the multitude of effects that accompany this dementia.
Pharmacological and receptor-ligand binding studies of the cloned orphanin FQ (OFQ) receptor suggest that multiple forms of this receptor may exist. To further characterize the OFQ receptor (OFQR), we attempted to isolate the gene encoding this receptor in rat. The OFQR gene exceeds 10 kb in length and contains six exons ranging from 34 to 524 bp that are interrupted by five introns. The ATG translation initiation codon is located in exon 2, and the open reading frame consists of 1283 bp. Primer extension analysis of the gene revealed two major transcription initiation sites: one in the 5' flanking region and the other in intron 1. The rat OFQR gene appeared to be alternatively spliced to yield multiple mRNAs. Four splice variants deleted for exon 1 were expressed only in brain. In contrast, five isoforms containing exon 1 were expressed in various tissues, such as brain, testes, and gastrointestinal tract. These data suggest that unique regions in intron 1 and in the 5' flanking region of the OFQR gene contribute to the regulation of its expression in different tissues.
Orphanin FQ/nociceptin (OFQ/N) has been shown to modulate nociception, responses to stress and anxiety. We investigated OFQ/N function in human immune cells. We find that monocytic U937, T lymphocytic CEM, and MOLT-4 cell lines express OFQ/N binding sites at levels comparable to that of human SH-SY5Y neuroblastoma cells. We show that OFQ/N receptors are functionally coupled to G proteins in these cells. Finally OFQ/N decreases proliferation of phytohemagglutinin-stimulated peripheral blood mononuclear cells in vitro at doses ranging from 10(-13) to 10(-8) M. Thus, our data suggest that OFQ/N and OFQ/N receptor may act as an immunomodulatory system.
Until recently the opioid receptor family was thought to consist of only the mu-, delta- and kappa-receptors. The cloning of opioid receptor like receptor (ORL1) and its endogenous ligand nociceptin/orphanin FQ, which displayed anti-opioid properties, has raised the issue of functional co-operativity of this system with the classical opioid system. ORL1 receptor knockout mice have been successfully developed by homologous recombination to allow the issue of potential heterogeneity of this receptor and also of compensatory changes in mu-, delta- or kappa-receptors in the absence of ORL1 to be addressed. We have carried out quantitative autoradiographic mapping of these receptors in the brains of mice that are wild-type, heterozygous and homozygous for the deletion of the ORL1 receptor. ORL1, mu-, delta- and kappa-receptors were labelled with [(3)H] leucyl-nociceptin (0.4 nM), [(3)H] DAMGO (4 nM), [(3)H] deltorphin-I (7 nM), and [(3)H] CI-977 (2.5 nM) respectively. An approximately 50% decrease in [(3)H] leucyl-nociceptin binding was seen in heterozygous ORL1 mutant mice and there was a complete absence of binding in homozygous brains indicating the single gene encodes for the ORL1 receptor and any putative subtypes. No significant gross changes in the binding to other opioid receptors were seen across genotypes in the ORL1 mutant mice demonstrating a lack of major compensation of classical opioid receptors in the absence of ORL1. There were a small number of region specific changes in the expression of classical opioid receptors that may relate to interdependent function with ORL1.
The isolation of an opioid receptor-related clone soon led to the isolation and characterization of a new neuropeptide, termed orphanin FQ or nociceptin (OFQ/N). This heptadecapeptide binds to the NOP(1) (previously termed ORL1) receptor with exceedingly high affinity, but does not interact directly with classical opioid receptors. Functionally, the actions of OFQ/N are diverse and intriguing. Most work has focused upon pain mechanisms, where OFQ/N has potent anti-analgesic actions supraspinally and analgesic actions spinally. Other OFQ/N activities are less clear. The diversity of responses might reflect NOP(1) receptor heterogeneity, but this remains to be established. The actions of this neurochemical system may also be uniquely dependent on contextual factors, both genetic and environmental. This review will address the molecular biology and behavioral pharmacology of OFQ/N and its receptor.
The orphanin peptide system, although structurally similar to the endogenous opioid family of peptides and receptors, has been established as a distinct neurochemical entity. The distribution of the opioid receptor-like (ORL1) receptor and its endogenous ligand orphanin FQ (OFQ) in the central nervous system of the adult rat has been recently reported, and although diffusely disseminated throughout the brain, this neuropeptide system is particularly expressed within stress and pain circuitry. Little is known concerning the normal expression of the orphanin system during gestation, nor how opiate or stress exposure may influence its development. Using in situ hybridization techniques, the present study was undertaken to determine the normal pattern of expression of ORL1 mRNA in the human and rat brain at various developmental stages. Rat embryos, postnatal rat brains and postmortem human brains were collected, frozen and cut into 15 microm coronal sections. In situ hybridization was performed using riboprobes generated from cDNA containing representative human and rat ORL1 and OFQ sequences. Both ORL1 and OFQ mRNA is detected as early as E12 in the cortical plate, basal forebrain, brainstem and spinal cord. Expression for both ORL1 and OFQ is strongest during the early postnatal period, remaining strong in the spinal cord, brainstem, ventral forebrain, and neocortex into the adult. Human ORL1 and OFQ expression is observed at 16 weeks gestation, remaining relatively unchanged up to 36 weeks. The influence of early orphanin expression on maturation of stress and pain circuitry in the developing brain remains unknown.
Although orphanin FQ/nociceptin (OFQ/N) receptors are a member of the opioid receptor family of receptors, they bind traditional opioids with very poor affinity. We now demonstrate that mu opioid receptors can physically associate with OFQ/N receptors, resulting in a complex with a unique binding selectivity profile. Immunoprecipitation of epitope-tagged OFQ/N receptors co-precipitates mu receptors. When the two receptors were co-expressed in CHO cells, [3H]OFQ/N retained its high binding affinity for its receptor. However, co-expression of the two receptors increased by up to 250-fold the affinity of a series of opioids in [3H]OFQ/N binding assays. This enhanced affinity was limited to agonists with high affinity for mu receptors. Selective kappa(1) and delta opioids did not lower binding. Despite the dramatic increase in affinity for the opioid agonists in co-expressing cells, the opioid antagonists naloxone and diprenorphine failed to compete [3H]OFQ/N binding.
Nociceptin/orphanin FQ (N/OFQ) is a recently identified neuropeptide that has been implicated in a multitude of CNS functions. These include nociception, feeding, cognition, locomotion, stress and neuroendocrine control. The endogenous receptor for this ligand is the nociceptin/orphanin FQ peptide (NOP) receptor. The distribution of NOP in rodent has been widely reported by the use of in situ hybridization, immunohistochemistry and autoradiographic radioligand binding but less is known of its localization in higher species. We have therefore sought to optimize and determine the distribution of (125)I[Tyr(14)]N/OFQ binding sites in macaque primate brain and spinal cord. Highest levels of binding were observed in neocortical areas, hippocampus, amygdala, caudate nucleus and putamen, medial thalamic nuclei and superficial laminae of the superior colliculus. These novel data present for the first time, the distribution of N/OFQ receptors in non-human primate CNS and, by comparison with localization in the rat, reveal that species differences may exist in the distribution of this neuropeptide receptor. These data have important implications regarding the roles of N/OFQ across species and may have ramifications in the interpretation of preclinical pharmacological studies.
Identification of a high affinity orphanin FQ
- Mathis Jp Ie Goldberg
- Letchworth
- Ryan-Moro J Sr
- Pasternak
Mathis JP, Goldberg IE, Letchworth SR, Ryan-Moro J, Pasternak GW (1999) Identification of a high affinity orphanin FQ/nociceptin(1–11)
Autoradiographic localization of 125 I[Tyr 14
- Letchworth
- Mathis Jp Sr
- Rossi Gc Rj Bodnar
- Pasternak
Letchworth SR, Mathis JP, Rossi GC, Bodnar RJ, Pasternak GW (2000) Autoradiographic localization of 125 I[Tyr 14 ]orphanin FQ/no-ciceptin and