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

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

Scientific RepoRts | 5:11676 | DOi: 10.1038/srep11676 neuropeptides contribute to different sensory modalities in primates. As dorsal horn neurons receive sensory information from primary afferents that innervate a large area of dermatomes and deeper tissues of the body 10 , it is pivotal to know the functional evidence of opioid-related neuropeptides with different binding affinities to MOP, KOP, DOP, and NOP receptors in regulating itch and pain in the spinal cord of primates.
In the past few years, there have been several exciting advances in identifying non-opioid neuropeptides, cells and neural circuits centrally in rodent models of itch 11,12 . One of the central itch mediators is the gastrin-releasing peptide (GRP) and its cognate receptor (BB 2 receptor) 13 . GRP has been used to elicit scratching activity in rodents 14 . Using BB 2 receptor mutant mice, an elegant study demonstrated that BB 2 receptors selectively mediate itch scratching, but not pain behavior, in the spinal cord of mice 15 . Moreover, BB 2 receptors are highly expressed in the spinal cord of monkeys displaying excessive scratching 16 and serum GRP levels in patients with atopic dermatitis are higher than those in healthy human subjects 17 . These recent findings pinpoint the key role of the GRP-BB 2 receptor system in mediating itch sensation. However, there is no functional study to investigate the basic characteristics such as the magnitude and duration of itch-eliciting effects of spinally delivered GRP as compared to MOP receptor-mediated itch, a well-documented phenomenon in the clinical setting 9 , and to determine how differently both BB 2 and MOP receptors regulate itch and pain in primates.
There is now an extensive body of literature documenting that the anatomical, neurochemical, and neuropharmacological aspects of receptors are similar between nonhuman primates and humans [18][19][20] . Conversely, the translational potential of ligands and targets discovered in rodents to primates remains to be established. For example, the transient receptor potential vanilloid type1 (TRPV1)-expressing neurons are demonstrated to mediate different itch-eliciting actions in mice 21 . However, a recent clinical trial concluded that a TRPV1 antagonist SB705498 is unlikely to be of symptomatic benefit for histaminergic and non-histaminergic itch 22 . Furthermore, rodents do not display robust scratching responses when they are injected spinally with morphine, which is known to induce itch in humans 23,24 . In contrast, spinal administration of morphine produces analgesia and itch simultaneously in both nonhuman primates and humans 7,9 . Therefore, the nonhuman primate model of spinally elicited itch serves as a translational bridge which not only validates drugs for ameliorating morphine-induced itch 25,26 , but also identifies promising ligands which produce morphine-like analgesia without eliciting itch sensation 27,28 .
Given that intrathecal delivery procedure in nonhuman primates has been established to provide the sound evidence of cause and effect for diverse ligands regulating itch and pain 8,27,28 , it is important to conduct pharmacological studies in awake behaving nonhuman primates to study neuropeptides and their cognate receptors. Therefore, the aim of this study was to address a longstanding, yet unanswered, fundamental question, namely, what are the functional consequences of neuropeptides in regulating itch and pain in the spinal cord of primates? In particular, we conducted a large-scale, translational nonhuman primate study to determine (1) effects of opioid-related neuropeptides and GRP on eliciting itch scratching and inhibiting inflammatory pain, (2) selective versus general effectiveness of MOP and BB 2 receptor antagonists as anti-itch agents, and (3) the functional role of the dynorphin A-KOP receptor system in itch and pain processing.
To side-by-side compare the itch-eliciting effects of β -endorphin and GRP, we determined the dose-responses for the magnitude and duration of both peptide-elicited scratching in terms of scratching responses, scratching time, and the ratios between body and head scratches in the second group of monkeys (n = 6).     (Fig. 3I,J). We further determined the potential antihyperalgesic effects of both peptides in the assay of carrageenan-induced hyperalgesia. Intrathecal β -endorphin 100 nmol significantly attenuated carrageenan-induced hyperalgesia and its antihyperalgesic effects lasted for 2 hours [F(2,10) = 265.5; p < 0.05] (Fig. 4A). In contrast, GRP 10 nmol did not produce antihyperalgesic effects (Fig. 4B).

Discussion
Both itch and pain are unpleasant sensory experiences accompanied with different behavioral responses. By using nonhuman primate behavioral assays, this study is the first to define the functional roles of diverse opioid-related neuropeptides and GRP in regulating itch and pain in the spinal cord of primates. Four major novel findings are reported herein. First, opioid-related neuropeptides, depending on their receptor selectivity and efficacy, can differentially elicit itch scratching and ameliorate inflammatory pain. Second, both β -endorphin and GRP elicited similar magnitude and duration of robust scratching responses; unlike β -endorphin regulating both itch and pain, GRP only elicits itch sensation. Third, both spinal MOP and BB 2 receptors can independently modulate itch. Fourth, without affecting  It is known that synthetic MOP receptor agonists produce analgesia, but they elicit itch in both nonhuman primates and humans 9,31 . In this study, there are different degrees of itch-eliciting and pain-inhibiting effects between endomorphins and β -endorphin. The [ 35 S]GTPγ S binding stimulation characterized endomorphins and β -endorphin as low and high efficacy agonists at MOP receptors, respectively 32 . Both endomorphin-1 and endomorphin-2 elicited mild-to-moderate scratching responses and produced partial antihyperalgesia. In contrast, β -endorphin produced robust scratching and full antihyperalgesia. This difference illustrates a correlation between the in vitro ligand efficacy and the magnitude of ligand effects elicited in vivo 33 . Interestingly, these efficacy-dependent MOP receptor-mediated itch-eliciting effects can be observed in monkeys 25,34 , but not in mice 35 . For example, DAMGO, a widely used control ligand as a full MOP receptor agonist, has higher efficacy than morphine 32 . Intrathecal DAMGO elicited a greater magnitude of scratching (~1,200 scratches/15 min) than morphine (~600 scratches/15 min) in monkeys 34 . In contrast, intrathecal DAMGO and morphine both only elicited mild scratches (i.e., ~30 scratches/30 min) in mice 35 . This functional difference in itch scratching documents a species difference in the pharmacological actions of spinal MOP receptor-expressing neurons.
DOP and NOP receptor preferring neuropeptides did not significantly increase scratching responses, but they produced different degrees of antihyperalgesic effects. DOP receptor neuropeptides, met-enkephalin and leu-enkephalin, only produced transient partial antihyperalgesia. The DOP receptor mRNA level is relatively low or undetectable in the spinal cord of primates 36,37 , which may explain a minimal role of the enkephalin-DOP receptor system in pain processing in monkeys. In contrast, NOP receptor neuropeptide N/OFQ produced full antihyperalgesia which lasted for 1.5 hours. Based on a series of anatomical, neurobiological, and pharmacological studies, the spinal N/OFQ-NOP receptor system has been indicated to play a crucial role in pain processing of both rodents and primates 19,38,39 . Like MOP receptors, NOP receptors are coupled to Gi/Go proteins and activation of NOP receptor inhibits forskolin-stimulated cAMP production and calcium currents, activates potassium channels, and inhibits basal and stimulated release of various neurotransmitters including substance P 38 . More importantly, spinal NOP receptor agonists produce MOP receptor agonist-comparable antinociceptive effects in rodents and nonhuman primates under different pain modalities 19,38,39 , which facilitates the development of NOP receptor-related ligands as spinal analgesics without itch side effect. Co-localization of MOP and NOP receptor immunoreactivity in the superficial laminae of the rat spinal cord was not observed and both receptors were expressed predominantly on different fiber systems 40 . If such anatomical evidence can be established in the dorsal horn neurons of primates, it may explain distinct pharmacological actions of MOP and NOP receptor agonists.
Interestingly, both β -endorphin and GRP elicited robust scratching responses. Unlike MOP receptor agonist-elicited itch in rodents as mentioned above, GRP is a neuropeptide which can centrally elicit robust scratching activity in both rodents and primates 24,41 . The magnitude and duration of scratching elicited by both neuropeptides are similar based on their scratching number, time, and location in monkeys. To our knowledge, GRP is the first non-opioid peptide identified in the spinal cord of primates which can elicit MOP receptor agonist-comparable scratching activity. However, unlike MOP receptor agonists including β -endorphin, GRP did not produce antihyperalgesic effects. These findings together validate the translatability of somatosensory function of spinal GRP from mice 15 to monkeys and conclude that spinal GRP selectively elicits itch with a minimum role in regulating pain in primates. Some cholestatic patients experienced itch and analgesia and their symptoms responded to opioid receptor antagonists that can ameliorate itch but cause opioid-like withdrawal discomfort 2,4 . Based on functional evidence of opioid-related neuropeptides in primates, β -endorphin but not endomorphins and enkephalins, could be the key neuropeptide for mediating such effects. As β -endorphin and GRP are two key neuropeptides eliciting robust scratching activity in primates, it will be important to compare both peptide levels in the cerebrospinal fluid of different populations of patients suffering from chronic itch.
Selective MOP and BB 2 receptor antagonists, naltrexone and RC-3095, dose-dependently attenuated β -endorphin-and GRP-elicited robust scratching, respectively. However, a functionally MOP receptor-selective dose of naltrexone 30 nmol did not block GRP-induced scratching. The opposite was true for RC-3095. Effects of both antagonists provide pharmacological evidence of the functional selectivity indicating that the spinal β -endorphin-MOP receptor and GRP-BB 2 receptor systems independently mediate itch in primates. Although intrathecal morphine-induced mild and transient scratching could be inhibited by a BB 2 receptor antagonist 35 , the present study does not support the notion that the BB 2 receptor is required for opioid-induced itch 35 . Both MOP and BB 2 receptor mRNA are present in the spinal cord of primates 16,36,42 . It is important to further investigate whether there are two distinct populations of dorsal horn neurons, each expressing either MOP or BB 2 receptors, in primates. More importantly, both MOP and BB 2 receptors are the only two receptor systems identified so far for mediating robust itch scratching in primates and supported by well-grounded pharmacological evidence (i.e., agonists elicit itch which can be blocked by corresponding receptor antagonists) 25,34 . Future development of mechanism-based antipruritics may use both spinal MOP and BB 2 receptor systems as a pharmacological basis to design bifunctional MOP-BB 2 receptor antagonists as potential antipruritics.
Another intriguing finding is that KOP receptor neuropeptide dynorphin A(1-17) attenuated both β -endorphin-and GRP-elicited robust scratching without affecting pain processing and the anti-itch effects of dynorphin A could be reversed by a KOP receptor antagonist. Although reduced activity of the endogenous dynorphin A-KOP receptor system has been implicated for the increased scratching activity 43,44 , there is no direct functional evidence in terms of behavioral effects of dynorphin A on scratching responses. The present study is the first to provide the functional consequence of spinal KOP receptor activation by dynorphin A. The anti-itch effect of KOP receptor agonists was first identified in the mid-1980s 45 . There have been some rodent and nonhuman primate studies, indicating that KOP receptor agonists with different chemical structures have a broad application as antipruritics against scratching responses elicited by diverse pruritogens 8,30,43 . In particular, low doses of KOP receptor agonists are effective for inhibiting scratching and higher doses of KOP receptor agonists are required to produce antinociceptive effects which are associated with sedation 8,30,46,47 . These early findings have led to the development of a KOP receptor agonist, nalfurafine, which is effective in treating patients with uremic pruritus 48 . Interestingly, a recent study has identified specific inhibitory interneurons, B5-I neurons, which express dynorphin A. Acute inhibition of B5-I neurons increased scratching and KOP receptor agonists and antagonists can decrease and increase scratching, respectively in the mouse spinal cord 44 . It will be valuable to further investigate whether patients with chronic itch have a lower level of dynorphin A in the cerebrospinal fluid.
In summary, by examining both behavioral and pharmacological factors, this study elucidates the functional roles of opioid-related neuropeptides and GRP in regulating itch and pain in the spinal cord of primates. Only activating spinal MOP or BB 2 receptors can independently elicit profound scratching activity. The potential up-regulation of the β -endorphin-MOP receptor and/or GRP-BB 2 receptor systems and down-regulation of the dynorphin A-KOP receptor system may intertwine in patients with chronic itch. MOP and NOP receptor preferring neuropeptides may be the key mediators for inhibiting pain processing in individuals under inflammatory pain. This translational nonhuman primate behavioral model with spinal delivery of ligands bridges a scientific gap in the functional roles of neuropeptides in primates, provides physiological relevance to patients with changed sensory modalities, and facilitates transforming discoveries of novel ligand-receptor systems into future therapies in humans.

Methods
Subjects. Eighteen adult male and female rhesus monkeys (Macaca mulatta) weighing between 6.1 to 13.4 kg were used. These monkeys were individually housed. Their daily diet consisted of approximately 25 to 30 biscuits (Purina Monkey Chow; Ralston Purina Co., St. Louis, MO, USA), fresh fruit, and water ad libitum. All monkeys had been previously trained in the warm water tail-withdrawal assay and acclimated to being video-recorded in-cage. They were housed in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The study protocols were approved by the Animal Care and Use Committee at the University of Michigan (Ann Arbor, MI, USA) and Wake Forest University (Winston-Salem, NC, USA). All animal care and experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health (Bethesda, MD, USA).

Procedures. Itch Scratching Responses.
Monkeys were recorded in their home cages in order to evaluate if they displayed increasing scratching behavior, which has been demonstrated to be associated with an itch sensation 7,34 . The quantification of scratching is described in SI Methods.
Nociceptive Responses. The warm water tail-withdrawal latency in 46 °C water after the carrageenan administration 31 was used to measure the antihyperalgesic effects of neuropeptides. Briefly, carrageenan (2 mg) was administered subcutaneously in the monkey's tail to elicit hyperalgesic responses. The measurement of antihyperalgesia is described in SI Methods.
Data Analysis. Mean values (mean ± SEM) were calculated from individual values for all behavioral endpoints. Comparisons were made for the same monkeys across all test sessions in the same experiment. Individual tail-withdrawal latencies were converted to the percentage of maximum possible antihyperalgesic effects, as defined in SI Methods.
Drugs. Naltrexone HCl, opioid-related neuropeptides (National Institute on Drug Abuse, Bethesda, MD, USA), GRP, nor-binaltorphimine HCl (Tocris Bioscience, Minneapolis, MN, USA) and RC-3095 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in sterile water. The neuropeptide alone or combined with the antagonist was delivered intrathecally at a total volume of 1 mL. A detailed description of the lumbar intrathecal drug delivery has been previously described 8,34 . The neuropeptide was delivered intrathecally with a 10-day inter-injection interval as previous studies did 8,25 .