Nociceptin/orphanin FQ metabolism and bioactive metabolites

doi:10.1016/S0196-9781(00)00228-X

Peptides

Volume 21, Issue 7, July 2000, Pages 919-922

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Abstract

The endogenous ligand for the orphan NOR receptor (earlier named ORL1) was recently discovered. This ligand, nociceptin/orphanin FQ is involved in a number of pharmacological actions in the CNS, including modulation of pain and cognition. However, its specific physiological role remains to be determined. Two major pathways of metabolism have been identified; the action of aminopeptidase(s) that prominently occurs in plasma, and endopeptidase activity that successively generates the N-terminal 1–13 and 1–9 fragments. Both pathways result in fragments that are inactive at the NOR receptor. However, short N-terminal fragments appear to be active in blocking the release of substance P from primary afferent C-fiber terminals in the dorsal spinal cord. The same endopeptidase(s) may also be involved in the fragmentation of dynorphin A since the inhibitor profile is similar. Enzyme activity is upregulated by morphine using either peptide as substrate that may lead to pharmacological interactions.

Introduction

Peptide metabolism, defined as the cleavage of a peptide bond, is an important area of study for a variety of reasons. It is obvious that metabolic fragmentation may lead to inactivation. Definition of inactivation pathways allows the design of enzyme inhibitors that may be of pharmacological interest. A now classic example is endopeptidase 24.11 (“enkephalinase”), that generates inactive metabolites from enkephalins, and its inhibitors that are analgesic and substitute in opiate abstinence [20]. Metabolism may also generate products that maintain activity but are less potent, or more interestingly, molecules with another activity. Inhibitors of such transformations may also be pharmacologically relevant. A prominent example is the ACE-inhibitors that block the activity of angiotensin converting enzyme (ACE). These examples indicate that at least in certain cases it is possible to design inhibitors of peptide metabolism of pharmacological relevance, indirectly showing that enzymatic transformations can be selective. In experimental studies, enzyme inhibitors can be used to investigate the physiological function of a particular peptide.

Nociceptin/orphanin FQ (N/OFQ), a 17-amino acid peptide has been defined as a major product from the pronociceptin protein and as the natural ligand for the NOR receptor [7], [13], [19]. All available data suggests that N/OFQ is also the endogenous ligand for this receptor [4], [5], [18]. The partial sequence homology with dynorphins and particularly dynorphin A suggests a common evolutionary origin of the prodynorphin and pronociceptin genes and raises the possibility that the same or similar metabolic enzymes are relevant for both N/OFQ and dynorphin A, a possibility to be considered here.

N/OFQ metabolism has been studied in different tissues (Table 1). It will obviously make a difference whether experimental studies are done in vivo or in vitro with brain slices, dissociated neurons or subcellular fractions of brain tissue. If peptides are given systemically for experimental or clinical purposes, metabolism in blood is going to be a significant factor as well. This review of the current literature will emphasize the experimental conditions used for the studies.

Section snippets

Generation of N/OFQ through pronociceptin metabolism

The precursor of N/OFQ, pronociceptin has recently been shown to be metabolized in amygdala and hypothalamus by the processing enzyme, PC2, an enzyme belonging to the subtilisin-like proprotein convertase family. Pintar et al. [17] showed that the production of N/OFQ (pronociceptin 141–157) was markedly reduced (60–90%) in PC2-knockout mice. The enzyme also seems to split pronociceptin 160–187 into two parts, one of which is the recently discovered nocII peptide (pronociceptin 160–176). The

N/OFQ and N/OFQ fragments occurring naturally

The wide distribution of N/OFQ and the NOR receptor throughout the CNS indicates a role for this peptide not only in nociception but also in cognition, motor control, sensory processing and a multitude of other physiological functions [1], [15]. However, our knowledge about naturally occurring N/OFQ fragments is very limited so far.

N/OFQ metabolism in vivo after microinjection into rat brain structures

The in vivo metabolism of N/OFQ in rat hippocampus has been studied using size-exclusion chromatography linked to electrospray ionization mass spectrometry [27]. This method is rapid, sensitive and unambiguous and provides a good insight into in vivo metabolic patterns. Rats with bilateral cannulas in the dorsal hippocampus were given a dose of N/OFQ (10 nmol/rat) or artificial CSF directly into the CA3-region of the hippocampus. After various time points (0–2 h) the hippocampus was dissected,

N/OFQ metabolism in mouse brain slices and in mouse brain in vivo

The metabolism of N/OFQ in mice has recently been reported. Mouse brain cortical slices were incubated with N/OFQ in the presence or in the absence of various peptidase inhibitors. N/OFQ was cleaved at the Phe¹-Gly², Ala⁷-Arg⁸, Ala¹¹-Arg¹², and Arg¹²- Lys¹³ bonds, forming four main metabolites, N/OFQ (2–17), (1–12), (1–11), and (1–7) [14]. The enzymes responsible were found to be aminopeptidase N and endopeptidase 24.15 and were completely blocked by selective enzyme inhibitors. Endopeptidase

N/OFQ metabolism in spinal cord

The metabolism of N/OFQ in rat spinal cord homogenate has recently been studied using mass spectrometry [28]. N/OFQ was incubated with rat spinal cord homogenate for various time periods and analyzed for fragmentation. It was metabolized mainly into N/OFQ(1–11) that was further truncated into N/OFQ(1–6) as a final product. The enzyme responsible appeared to be a soluble, neutral serine endopeptidase, dependent on metal ions.

The N/OFQ(1–6) fragment was shown to have a biphasic effect in various

N/OFQ metabolism in cell culture—effect of morphine and comparison with dynorphin metabolism

To reduce the complexity of brain tissue and the number of reciprocal interactions, metabolic studies have been run in cultures of rat brain primary cortical cells, human SH-SY5Y neuroblastoma and U1690 small cell lung carcinoma. All these cell systems were previously found to be able to metabolize dynorphin peptides [30]. The fragmentation of N/OFQ was qualitatively the same in all cell lines, with N1–13 and N1–9 as the main fragments. The cleavage was inhibited by metals and SH-reagents

N/OFQ metabolism in human plasma

N/OFQ metabolism in human plasma has been studied using mass spectrometry to identify peptide fragments [33]. Like other peptides, N/OFQ is degraded by aminopeptidases in plasma. The main fragment found was (2–17) that was further metabolized into N/OFQ(3–17), (4–17), and (5–17). However, N/OFQ seemed to be more resistant to degradation than dynorphin A. These transformations eliminate activity at the NOR receptor. Small amounts of other fragments were also found, including N/OFQ(1–8), (1–10),

Pharmacologic activity of N/OFQ fragments

N-terminal truncation leads to complete inactivity at the NOR receptor and C-terminal fragments have not been reported to have activity in any system. N-terminal fragments are successively less active with N1–13NH₂ being the minimum active sequence at the NOR receptor [4]. However, the nonamidated variant, N1–13, does not retain any affinity [5]. It has previously been shown that N/OFQ severely impairs spatial learning in the Morris Water Task at a dose of 10 nmol/rat [26]. However, the 1–13

Conclusion

It has to be pointed out that there are considerable gaps in our knowledge on N/OFQ metabolism regarding species/tissue/experimental systems etc. Table 1 summarizes the findings obtained so far.

Truncation of a few amino acids from the C-terminus of N/OFQ does not terminate affinity at the NOR receptor. However, the major fragments produced in brain, N1–13 and N1–9 have no appreciable affinity. These transformations may be generated by the same enzymes that also cleave dynorphin A. Truncation of

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Nociceptin/orphanin FQ (N/OFQ) and N/OFQ peptide (NOP) receptor are expressed and distributed in various regions such as central nervous system (CNS), peripheral nervous system, immune system, and peripheral tissues. N/OFQ and NOP receptor have important roles on a variety of physiological, pathophysiological, regulatory, and dysregulatory mechanisms in the living body. Both activation and blockade of NOP receptor function have displayed clinical potential of NOP receptor agonists and antagonists for the treatment of various diseases or pathophysiological conditions, respectively. Potent and selective NOP receptor agonists/antagonists are also useful tools to investigate the various mechanisms mediated by NOP receptor–N/OFQ system. As the present study, a series of (4-arylpiperidine substituted-methyl)-[bicyclic (hetero)cycloalkanobenzene] analogs was designed, synthesized, and biologically evaluated in vitro to seek and identify potent and selective, small-molecules of nonpeptide NOP receptor antagonists, which resulted in the discovery of novel potent small-molecule 15 with high human NOP receptor selectivity over human μ receptor. The structure–activity relationship (SAR) of the potency and selectivity, structure–metabolic stability relationship (SMR), and SAR of hERG (human ether-a-go-go related gene) potassium ion channel binding affinity for the analogs in the present studies in vitro provided or suggested significant and/or useful structural determinants and insights for the respective purposes. The superior profiles of compound 15 are discussed with a viewpoint of multisite interactions between ligand and NOP receptor, together with the results of previous NOP receptor agonist/antagonist studies.
The biology of Nociceptin/Orphanin FQ (N/OFQ) related to obesity, stress, anxiety, mood, and drug dependence
2014, Pharmacology and Therapeutics
Citation Excerpt :
Experimental data from these preparations support a role for endogenous N/OFQ in regulating stress responses, development of morphine tolerance, and alterations in pain thresholds (Reinscheid & Civelli, 2002; Chung et al., 2006). In addition, several studies have explored the post-translational processing and enzymatic metabolism of the N/OFQ (for review see Terenius et al., 2000; Hallberg & Nyberg, 2003). Collectively, the use of these various research tools has created the opportunity to explore the impact of manipulations of N/OFQ system on physiological function and integrated disease-related functional correlates (Largent-Milnes & Vanderah, 2010).
Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that was deorphanized in 1995. The generation of specific agonists, antagonists and receptor deficient mice and rats has enabled progress in elucidating the biological functions of N/OFQ. Additionally, radio-imaging technologies have been advanced for investigation of this system in animals and humans. Together with traditional neurobehavioral techniques, these tools have been utilized to identify the biological significance of the N/OFQ system and its interacting partners. The present review focuses on the role of N/OFQ in the regulation of feeding, body weight homeostasis, stress, the stress-related psychiatric disorders of depression and anxiety, and in drug and alcohol dependence. Critical evaluation of the current scientific preclinical literature suggests that small molecule modulators of nociceptin opioid peptide receptors (NOP) might be useful in the treatment of diseases related to these biological functions. In particular, the literature data suggest that antagonism of NOP receptors will produce anti-obesity and antidepressant activities in humans. However, there are also contradictory data discussed. The current literature on the role of N/OFQ in anxiety and addiction, on the other hand points primarily to a role of agonist modulation being potentially therapeutic. Some drug-like molecules that function either as agonists or antagonists of NOP receptors have been optimized for human clinical study to test some of these hypotheses. The discovery of PET ligands for NOP receptors, combined with the pharmacological tools and burgeoning preclinical data set discussed here bodes well for a rapid advancement of clinical understanding and potential therapeutic benefit.
Acute and subchronic treatments with selective serotonin reuptake inhibitors increase Nociceptin/Orphanin FQ (NOP) receptor density in the rat dorsal raphe nucleus; Interactions between nociceptin/NOP system and serotonin
2013, Brain Research
Citation Excerpt :
Consequently, even if UFP-101 displays a very weak affinity for KOP receptors, the specificity of N/OFQ binding on NOP receptor has been established, leading to the conclusion that N/OFQ effect on extracellular 5-HT in the DRN could be attributed to its effect on NOP receptor. The short-lasting effect (within 60 min) could be attributed to the reversibility of N/OFQ binding on its receptor and to the inactivation of nociceptin in the DRN by enzymatic systems: aminopeptidase and endopeptidase (Terenius et al., 2000), and/or to diffusion processes. NOP receptor is well described as a metabotropic receptor coupled to a Gi protein inducing a decrease in cAMP formation (Hawes et al., 2000).
Nociceptin/Orphanin FQ is the endogenous ligand of NOP receptor, formerly referred to as the Opioid Receptor-Like 1 receptor. We have previously shown that NOP receptors were located on serotonergic neurons in the rat dorsal raphe nucleus, suggesting possible direct interactions between nociceptin and serotonin in this region, which is a target for antidepressant action. In the present study, we investigated further the link between Selective Serotonin Reuptake Inhibitor (SSRI) antidepressant treatments and the nociceptin/NOP receptor system. Intraperitoneal administration of the SSRI citalopram induced an increase in NOP-receptor density, measured by autoradiographic [³H] nociceptin binding, in the rat dorsal raphe nucleus, from the first to the 21st day of treatment. This effect was also observed with other SSRIs (sertraline, fluoxetine), but not with two tricyclic antidepressants (imipramine, clomipramine) and was abolished by pre-treatment with para-chlorophenylalanine, an inhibitor of serotonin synthesis. Using microdialysis experiments, we demonstrated that NOP-receptor activation by infusion of nociceptin 10⁻⁶ M or 10⁻⁵ M increased the level of extracellular serotonin in the dorsal raphe nucleus. This effect was abolished by co-infusion of the NOP-receptor antagonist UFP 101. These results confirm the existence of reciprocal interactions between serotonin and nociceptin/NOP transmissions in the dorsal raphe nucleus.
Sex differences in the Nociceptin/Orphanin FQ system in rat spinal cord following chronic morphine treatment
2012, Neuropharmacology
Nociceptin/Orphanin FQ (N/OFQ) appears to contribute to the development of morphine tolerance, as blockade of its actions will block or reverse the process. To better understand the contribution of N/OFQ to the development of morphine tolerance, this study examined the effect of chronic morphine treatment on levels of N/OFQ and levels and activity of the N/OFQ peptide (NOP) receptor in spinal cord (SC) from male and female rats. Both male and female Wistar rats showed less responsiveness to morphine after subcutaneous injection of escalating doses of morphine (10, 20, 40, 60 and 80 mg/kg, respectively) twice daily for five consecutive days. Male rats were more tolerant to the antinociceptive actions of morphine than females. The N/OFQ content of SC extracts was higher in females than in males, regardless of treatment; following chronic morphine treatment the difference in N/OFQ levels between males and females was more pronounced. N/OFQ content in cerebrospinal fluid (CSF) was reduced 40% in male and 16% in female rats with chronic morphine exposure, but increased in periaqueductal grey of both sexes. Chronic morphine treatment increased NOP receptor levels 173% in males and 137% in females, while decreasing affinity in both. Chronic morphine increased the efficacy of N/OFQ-stimulated [³⁵S]GTPγS binding to SC membranes from male rats, consistent with increased receptor levels. Taken together, these findings demonstrate sex differences in N/OFQ–NOP receptor expression and NOP receptor activity following chronic morphine treatment. They also suggest interplay between endogenous N/OFQ and chronic morphine treatment that results in nociceptive modulation.
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Leucine amino peptidase (LAP, EC 3.4.11.1) is a widely distributed metallo exopeptidase that catalyzes the hydrolysis of leucine residues from the amino-termini of protein or peptide substrates (Rawlings and Barrett, 2004). It has been the most extensively studied enzymes because it plays key role in the metabolism of biologically active peptides (Terenius et al., 2000; Goldberg et al., 2002) and has great potential for commercial applications (Kamphuis et al., 1992; Rao et al., 1998). Besides, LAP can improve flavor development (Toldra et al., 2000) and convert l-homophenylalanyl amide into l-homophenylalanine, the versatile intermediate for a class of angiotension 1 converting enzyme inhibitors, such as Enalapril, and Benzapril (Kamphuis et al., 1992).
Studies were conducted on the production of leucine amino peptidase (LAP) by Streptomyces gedanensis to ascertain the performance of the process in shake flask, parallel fermenter and 5-L fermenter utilizing soy bean meal as the carbon source. Experiments were conducted to analyze the effects of aeration and agitation rate on cell growth and LAP production. The results unveiled that an agitation rate of 300 rpm, 50% dissolved oxygen (DO) upholding and 0.15 vvm strategies were the optimal for the enzyme production, yielding 22.72 ± 0.11 IU/mL LAP in parallel fermenter which was comparable to flask level (24.65 ± 0.12 IU/mL LAP) fermentation. Further scale-up, in 5-L fermenter showed 50% DO and 1 vvm aeration rate was the best, producing optimum and the production was 20.09 ± 0.06 IU/mL LAP. The information obtained could be useful to design a strategy to improve a large-scale bioreactor cultivation of cells and production of LAP.
Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception
2011, Peptides
Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1–13), (1–11) and (1–7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3–1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1–13) and (1–11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1–13) and 4.75 for N/OFQ (1–11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1–7) led to be less potent than N/OFQ and its fragments, (1–13) and (1–11). Antinociception induced by N/OFQ or N/OFQ (1–13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1–13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1–11) and (1–7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1–13) still possess antinociceptive activity through NOP receptors.

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Regular paperNociceptin/orphanin FQ metabolism and bioactive metabolites

Abstract

Introduction

Section snippets

Generation of N/OFQ through pronociceptin metabolism

N/OFQ and N/OFQ fragments occurring naturally

N/OFQ metabolism in vivo after microinjection into rat brain structures

N/OFQ metabolism in mouse brain slices and in mouse brain in vivo

N/OFQ metabolism in spinal cord

N/OFQ metabolism in cell culture—effect of morphine and comparison with dynorphin metabolism

N/OFQ metabolism in human plasma

Pharmacologic activity of N/OFQ fragments

Conclusion

Brain Res

Eur J Pharmacol

Biochem Biophys Res Commun

Brain Res

Regul Pept

Biochem Biophys Res Commun

15 inhibitors potentiate behavioral effects mediated by nociceptin/orphanin FQ in mice. FEBS Lett

J Biol Chem

Brain Res

Brain Res

Neurosci Lett

Peptides

Brain Res

Brain Res

Brain Res

Peptides

Brain Res

Regular paper
Nociceptin/orphanin FQ metabolism and bioactive metabolites