Molecular and Epigenetic Aspects of Opioid Receptors in Drug Addiction and Pain Management in Sport.

Opioids are substances derived from opium (natural opioids). In its raw state, opium is a gummy latex extracted from Papaver somniferum. The use of opioids and their negative health consequences among people who use drugs have been studied. Today, opioids are still the most commonly used and effective analgesic treatments for severe pain, but their use and abuse causes detrimental side effects for health, including addiction, thus impacting the user's quality of life and causing overdose. The mesocorticolimbic dopaminergic circuitry represents the brain circuit mediating both natural rewards and the rewarding aspects of nearly all drugs of abuse, including opioids. Hence, understanding how opioids affect the function of dopaminergic circuitry may be useful for better knowledge of the process and to develop effective therapeutic strategies in addiction. The aim of this review was to summarize the main features of opioids and opioid receptors and focus on the molecular and upcoming epigenetic mechanisms leading to opioid addiction. Since synthetic opioids can be effective for pain management, their ability to induce addiction in athletes, with the risk of incurring doping, is also discussed.

A lesion-mimic mutant of Catharanthus roseus accumulates the opioid agonist, akuammicine.

Catharanthus roseus is a medicinal plant that produces an abundance of monoterpenoid indole alkaloids (MIAs), notably including the anticancer compounds vinblastine and vincristine. While the canonical pathway leading to these drugs has been resolved, the regulatory and catalytic mechanisms controlling many lateral branches of MIA biosynthesis remain largely unknown. Here, we describe an ethyl methanesulfonate (EMS) C. roseus mutant (M2-117523) that accumulates high levels of MIAs. The mutant exhibited stunted growth, partially chlorotic leaves, with deficiencies in chlorophyll biosynthesis, and a lesion-mimic phenotype. The lesions were sporadic and spontaneous, appearing after the first true bifoliate and continuing throughout development. The lesions are also the site of high concentrations of akuammicine, a minor constituent of wild type C. roseus leaves. In addition to akuammicine, the lesions were enriched in 25 other MIAs, resulting, in part, from a higher metabolic flux through the pathway. The unique metabolic shift was associated with significant upregulation of biosynthetic and regulatory genes involved in the MIA pathway, including the transcription factors WRKY1, CrMYC2, and ORCA2, and the biosynthetic genes STR, GO, and Redox1. Following the lesion-mimic mutant (LMM) phenotype, the accumulation of akuammicine is jasmonate (JA)-inducible, suggesting a role in plant defence response. Akuammicine is medicinally significant, as a weak opioid agonist, with a preference for the κ-opioid receptor, and a potential anti-diabetic. Further study of akuammicine biosynthesis and regulation can guide plant and heterologous engineering for medicinal uses.

[Mechanisms of liver-soothing and mind-regulating acupuncture mediating opioid receptor participated in the treatment of migraine].

OBJECTIVE: To observe the effect of "Shugan Tiaoshen"(liver-soothing and mind-regulating) acupuncture on behavior reactions, opioid receptor expressions in the anterior cingulate cortex tissue and inflammatory factors in the serum in migraine rats, in order to explore its mechanism underlying improvement of migraine. METHODS: In the first part of this study, forty male Wistar rats were randomized into control, model, routine acupuncture and "Shugan Tiaoshen" acupuncture groups (n=10/group), and in the second part, other 40 more male Wistar rats were randomized into low, medium and high dosage of blocker of µopioid receptor (OPRM）CTOP5 and PBS groups (n=10/group, for validating the involvement of opioid receptor in the effect of "Shugan Tiaoshen"). The migraine model was established by subcutaneous injection of glyceryl trinitrate. Routine acupuncture was applied to "Baihui" (GV20) and bilateral"Fengchi" (GB20), and "Shugan Tiaoshen" acupuncture applied to GV20, and bila-teral GB20, "Neiguan" (PC6) and "Taichong" (LR3), with the needles retained for 30 min. Behavior responses (head scratching, tail biting, cage climbing and number of going there and back) were scaled. Serum IL-1ß, IL-6 and TNF-α were detected by ELISA, and the expression levels of opioid receptor µ, δ and κ (OPRM, OPRD, OPRK) mRNAs and proteins in the anterior cingulate cortex were detected by fluorescence quantitative PCR and Western blot separately. In the second part of this study, CTOP solution (5µL at concentrations of 20µg/µL,10µg/µL and 5µg/µL) or PBS was injected into the bilateral rostral portions of anterior cingulate cortex 30 min before every "Shugan Tiaoshen" acupuncture intervention, followed by observing the behavioral changes and assaying the contents of serum IL-1ß, IL-6 and TNF-α. RESULTS: After modeling, the behavioral score, serum IL-1ß, IL-6 and TNF-α contents were significantly increased in the model group relevant to the control group (P<0.05), and the beha-vioral score had no significant difference among the model and two acupuncture groups before intervention (P>0.05). Whereas the expression levels of OPRM, OPRD and OPRK mRNAs and proteins had a slight increase in the model group (P>0.05). After the intervention, the behavioral score, serum IL-1ß, IL-6 and TNF-α contents were significantly decreased and the expression levels of OPRM, OPRD and OPRK mRNAs (2.150, 1.066 and 0.805 folds in the "Shugan Tiaoshen" group) and proteins (2.273, 0.901 and 0.893 folds in the "Shugan Tiaoshen" group) notably up-regulated in both "Shugan Tiaoshen" and routine acupuncture groups relevant to the model group (P<0.01, P<0.05), showing that the biggest up-regulation of mRNA expression was OPRM. Comparison between two acupuncture groups showed that the behavioral score, and serum IL-1ß, IL-6 and TNF-α contents were significantly lower, and the expression levels of OPRM, OPRD and OPRK mRNAs and proteins obviously higher in the "Shugan Tiaoshen" group than those in the routine acupuncture group (P<0.01,P<0.05). Results of the second part of this study showed that after injection of antagonist CTOP of OPRM, the therapeutic effect of "Shugan Tiaoshen" acupuncture was weakened in the reduction of behavioral score and serum IL-1ß, IL-6 and TNF-α contents, being minimal, moderate and maximum in the high, medium and low dose of antagonist relevant to PBS in sequence (P<0.05, P<0.01). CONCLUSION: "Shugan Tiaoshen" acupuncture can mitigate pain in migraine rats, which may be associated with its function in up-regulating the expressions of opioid receptors (especially OPRM), and in inhibiting inflammatory reaction in the anterior cingulate cortex.

Label-free cell phenotypic study of opioid receptors and discovery of novel mu opioid ligands from natural products.

ETHNOPHARMACOLOGICAL RELEVANCE: Mu opioid receptor (MOR) is mainly a drug target for analgesia. Opioid-like agonists such as morphine have been clinically used for analgesia but have potential adverse effects. MOR antagonists have been demonstrated to alleviate these side effects. Plants (Carthamus tinctorius L, Cynanchum otophyllum C. K. Schneid., Coffea arabica L., Prinsepia utilis Royle and Lepidium meyenii Walp.) and Ganoderma fungi (Ganoderma hainanense J. D. Zhao, Ganoderma capense (Lloyd) Teng, Ganoderma cochlear (Blume et Nees) Bres., Ganoderma resinaceum Boud and Ganoderma applanatum (Pers.) Pat.) are traditional medicines with beneficial effects on immunoregulation, analgesia and the nervous system, but whether MORs are engaged in their effects remains unknown. AIM OF THE STUDY: This work aimed to identify MOR ligands among compounds isolated from the above-mentioned 10 species, and to investigate selectivity against four opioid receptor subtypes. By analyzing the structure-activity relationship and off-target effects, we could provide a new direction for the future development of MOR drugs. MATERIALS AND METHODS: Four opioid receptor subtype models, including MOR, delta (DOR), kappa (KOR) and nop (NOR), were established with a label-free phenotypic dynamic mass redistribution assay to systematically profile the pharmacological properties of known ligands. Then, 82 natural compounds derived from the 10 species were screened against MOR to identify new ligands. The selectivity of the new ligands was characterized against the four subtypes, and off-target effects were also investigated on eight G protein-coupled receptors (GPCRs). RESULTS: The pharmacological properties of known ligands on transfected HEK293T-MOR, HEK293-DOR, HEK293-KOR and HEK293-NOR cell lines were characterized. Seven compounds purified from Ganoderma cochlear (Blume et Nees) Bres. and Carthamus tinctorius L were MOR antagonists with micromolar potency. Among them, compound 35 showed the strongest antagonistic activity on MOR with an IC50 value of 10.0 ± 3.0 µM. To a certain extent, these seven new antagonists, exhibited antagonistic activity on the other opioid receptor subtypes, and they had almost no effect on other GPCRs, including CB1, CB2, M2 and beta2AR. Additionally, a compound from Lepidium meyenii Walp. displayed MOR agonistic activity. CONCLUSIONS: The established screening models opened new avenues for the discovery and evaluation of opioid receptor ligand selectivity. Together, the novel MOR antagonists and agonists will enrich the inventory of MOR ligands and benefit related therapies.

Modulation of COX-2, INF-É£, glutamatergic and opioid systems contributes to antinociceptive, anti-inflammatory and anti-hyperalgesic effects of bis(3-amino-2-pyridine) diselenide.

Preclinical assays play a key role in research in research on the neurobiology of pain and the development of novel analgesics. Drugs available for the treatment of inflammatory pain are not fully effective and show adverse effects. Thus, we investigated the antinociceptive, anti-inflammatory and anti-hyperalgesic effects of bis(3-amino-2-pyridine) diselenide (BAPD), a new analgesic drug prototype. BAPD effects were investigated using nociception models induced by chemical (glutamate), immunologic (Freund's Complete Adjuvant - CFA) and thermal stimuli in Swiss mice. Mice were orally (p.o.) treated with BAPD (0.1-50 mg/kg) 30 min prior to the glutamate and hot-plate tests and a time-course (0.5 up to 8 h) of the antinociceptive effect of BAPD (50 mg/kg, p. o.) was evaluated in a CFA model. In the CFA model, BAPD effects on cyclooxygenase-2 (COX-2), tumor necrosis factor (TNFα) and interferon-γ (INF-γ) expression, myeloperoxidase (MPO) activity, oxidative (2,2'-Azino-bis-3-ethylbenzothiazoline 6-sulfonic acid and 2,2-diphe- nyl-1-picrylhydrazyl levels) and histological parameters were evaluated. The safety of the compound (50 and 300 mg/kg, p. o.) was verified for 72 h. BAPD reduced the licking time induced by glutamate and caused an increase in latency response to thermal stimulus. Naloxone reversed the antinociceptive effect of BAPD. Paw edema formation induced by glutamate or CFA injection was reduced by BAPD. Mechanical hyperalgesia induced by CFA was attenuated by BAPD. BAPD did not protect against the increase in MPO activity and decrease of the 2,2'-Azino-bis-3-ethylbenzothiazoline 6-sulfonic acid and 2,2-diphe- nyl-1-picrylhydrazyl levels induced by CFA. BAPD protected against histological alterations and reduction on the levels of gene expression COX-2 and INF-γ in the paw of mice exposed to CFA. BAPD was safe at the doses and time evaluated. BAPD exerts acute antinociceptive, anti-inflammatory and anti-hyperalgesic actions, suggesting that it may represent an alternative in the future development of new therapeutic strategies.

Maternal manipulation during late gestation (GDs 17-20) enhances ethanol consumption and promotes changes and opioid mRNA expression in infant rats.

Fetal ethanol experience generates learning and memories capable to increase ethanol consummatory behaviors during infancy. Opioid system seems to be involved in mediating those alcohol-related behaviors. In this work, we proposed to study the impact of prenatal exposure to a moderate ethanol dose, upon ingestion of the drug and possible ethanol-induced molecular changes on opioid precursor peptides (POMC, Pro-enk and Pro-DYN) and receptors (MOR, DOR and KOR) mRNA expression, in hypothalamus. Pregnant rats received during gestational days (GDs) 17-20, a daily intragastric (i.g.) administration with 2g/kg ethanol or water. A third group of dams was left undisturbed during pregnancy (Unmanipulated group). Intake test was conducted at postnatal days (PDs) 14-15. Three groups of pups were performed: control (no intake test), water (vehicle) and 5% ethanol. At the end of intake test blood samples were taken to quantify blood ethanol concentrations (BECs) and hypothalamus sections were obtained to perform qRT-PRC assessment of opioid precursor peptides and receptors. The analysis of the consummatory responses (% of consumption) and pharmacokinetic profiles (BECs) suggested that maternal manipulation induced by i.g. intubations, during the last four days of gestation (whenever ethanol or water), are sufficient to induce infantile ethanol intake during infancy. Gene expression from the hypothalamus of unmanipulated group revealed that infantile ingestive experiences with ethanol can down-regulate expression of mRNA Pro-Dyn and up-regulate mRNA expression of MOR and KOR. Finally, MOR mRNA expression was attenuated by prenatal i.g. manipulation in pups exposed to 5% ethanol.

The standardized Withania somnifera Dunal root extract alters basal and morphine-induced opioid receptor gene expression changes in neuroblastoma cells.

BACKGROUND: Behavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine's analgesic effect; however, little is known about the underpinning molecular mechanism(s). In order to shed light on this issue in the present paper we explored whether WSE promotes alterations of µ (MOP) and nociceptin (NOP) opioid receptors gene expression in neuroblastoma SH-SY5Y cells. METHODS: A range of WSE concentrations was preliminarily tested to evaluate their effects on cell viability. Subsequently, the effects of 5 h exposure to WSE (0.25, 0.50 and 1.00 mg/ml), applied alone and in combination with morphine or naloxone, on MOP and NOP mRNA levels were investigated. RESULTS: Data analysis revealed that morphine decreased MOP and NOP receptor gene expression, whereas naloxone elicited their up-regulation. In addition, pre-treatment with naloxone prevented the morphine-elicited gene expression alterations. Interestingly, WSE was able to: a) alter MOP but not NOP gene expression; b) counteract, at its highest concentration, morphine-induced MOP down-regulation, and c) hamper naloxone-induced MOP and NOP up-regulation. CONCLUSION: Present in-vitro data disclose novel evidence about the ability of WSE to influence MOP and NOP opioid receptors gene expression in SH-SY5Y cells. Moreover, our findings suggest that the in-vivo modulation of morphine-mediated analgesia by WSE could be related to the hindering of morphine-elicited opioid receptors down-regulation here observed following WSE pre-treatment at its highest concentration.

N/OFQ system in brain areas of nerve-injured mice: its role in different aspects of neuropathic pain.

Several studies showed that chronic pain causes reorganization and functional alterations of supraspinal brain regions. The nociceptin-NOP receptor system is one of the major systems involved in pain control and much evidence also suggested its implication in stress, anxiety and depression. Therefore, we investigated the nociceptin-NOP system alterations in selected brain regions in a neuropathic pain murine model. Fourteen days after the common sciatic nerve ligature, polymerase chain reaction (PCR) analysis indicated a significant decrease of pronociceptin and NOP receptor mRNA levels in the thalamus; these alterations could contribute to the decrease of the thalamic inhibitory function reported in neuropathic pain condition. Nociceptin peptide and NOP mRNA increased in the anterior cingulate cortex (ACC) and not in the somatosensory cortex, suggesting a peculiar involvement of this system in pain regulating circuitry. Similarly to the ACC, an increase of nociceptin peptide levels was observed in the amygdala. Finally, the pronociceptin and NOP mRNAs decrease observed in the hypothalamus reflects the lack of hypothalamus-pituitary-adrenal axis activation, already reported in neuropathic pain models. Our data indicate that neuropathic pain conditions affect the supraspinal nociceptin-NOP system which is also altered in regions known to play a role in emotional aspects of pain.

Electroacupuncture Ameliorates Propofol-Induced Cognitive Impairment via an Opioid Receptor-Independent Mechanism.

While general anesthesia is known to induce cognitive deficits in elderly and pediatric patients, its influence on adults is less well-characterized. The present study was designed to evaluate the influence of propofol on the learning and memory of young adult rats, as well as the potential neuroprotective role of electroacupuncture (EA) in propofol-induced cognitive impairment. Intravenous anesthesia with propofol was administered to young adult male Sprague-Dawley (SD) rats for 6 h, and EA was administered three times before and after anesthesia. The Morris Water Maze (MWM) test was conducted to determine the rat's cognitive performance following the anesthesia treatment. Our results showed that propofol induced obvious cognitive impairment in young adult rats, which could be ameliorated by multiple EA treatments. Moreover, the decreased level of phosphorylated glycogen synthase kinase 3 ß (pGSK-3ß) in the CA1 region of the hippocampus accompanying the cognitive impairment was also reversed by EA treatment. Further experiments demonstrated that neither 2 nor 10 mg/kg (I.P.) naloxone blocked the effect of EA, indicating that the neuroprotective effect of EA on propofol-induced cognitive impairment was not mediated via the opioid receptors. The present study suggests that EA could ameliorate the cognitive impairment induced by prolonged anesthesia with propofol in young adult rats, which is likely associated with pGSK-3ß levels in the CA1 independently of opioid receptors. These findings imply that EA may be used as a potential neuroprotective therapy for post-operative cognitive dysfunction (POCD).

[Effect of Electroacupuncture Intervention on Expression of Pain Sensory and Affective Proce- ssing-related µ-opioid Receptor, etc. in the Amygdala in Chronic Neuropathy Pain Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of pain sensory and affective processing-related µ-opioid receptor (MOR), glutamatergic AMPA receptor subunit GIuA 1, extracellular signal-regulated kinase (ERK 1/2), cAMP response element binding protein(CREB) in the amygdala in chronic constrictive injury (CC) + negative affection(NA) rats, so as to reveal its mechanism underlying pain relief. METHODS: A total of 32 male Wistar rats were randomized into normal control, model, EA, and anesthesia+ EA (AEA) groups (n = 8 in each group). The neuropathic pain NA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the paw-bottom in the pain-paired compartment. EA was applied to bilateral "Zusanli" (ST 36) and "Yanglingquan" (GB 34) for 30 min, once daily for 7 days. Thermal pain threshold (paw withdrawl latency, PWL) of the bilateral paws was measured by using a Tail-Flick Unit. The expression levels of MOR and p-CREB in the central amygdala (CeA) and those of MOR, GluA 1, p-ERK 1/2 and p-CREB in the right amygdala area were determined using immunofluorescence and Western blot, respectively. RESULTS: in comparison with the normal group, PWL difference (PWLD) values of the model group were significantly increased (P < 0.001), and the time spent in the CPA-paired. compartment was considerably decreased (P < 0.001). After EA, the PWLD levels of both EA and AEA groups were apparently decreased (P < 0.05), showing a pain relief; and the time spent in the CPA-paired compartment was apparently increased in the EA group (P < 0.05) , rather than in the AEA group (P > 0.05). Additionally , compared to the normal group, the expression level of MOR protein in the amygdala was remarkably increased (P < 0.05) and those of GIuA 1, p-ERK 2 and p-CREB proteins were apparently decreased (P < 0.05). After EA intervention for 7 days, the expression levels of these four proteins in the EA group, and those of MOR, p-ERK 2 and p-CREB in the AEA group were significantly up-regulated (P < 0.001, P < 0.01, P < 0.05). The expression level of GIuA 1 was significantly higher in the EA group than in the AEA group (P < 0.05). CONCLUSION: Repeated EA stimulation of ST 36-GB 34 has a definite effect in relieving both sensation and affection dimensions of pain in NA rats, which may be related to its effect in up-regulating the expression of GIuA 1 in the amygdala, but the effects of MOR, p-ERK 2 and p-CREB need being researched further.

Analgesic potential of standardized methanol stem bark extract of Ficus platyphylla in mice: Mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of the stem bark of Ficus platyphylla (FP) have been used in traditional the Nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. Previous studies have revealed the analgesic and anti-inflammatory effects of FP in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. PURPOSE/METHODS: In this study, we assessed the effects of the standardised extract of FP on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. We also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (COX) and 5-lipoxygenase (5-LO). RESULTS: FP (i) increased the hot plate nociceptive threshold and vocalisation threshold. The increase in hot plate nociceptive threshold was detectable over a period of 30min whereas the increase in vocalisation threshold persisted over a period of 90min. (ii) FP showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) FP inhibited the activities of COX-2 and 5-LO but not of COX-1. CONCLUSIONS: We provided evidence supporting the use of FP in Nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. It is interesting to note that the dual inhibition of COX-2 and 5-LO appears favourable in terms of both efficacy and side effect profile.

A new potent analgesic agent with reduced liability to produce morphine tolerance.

The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing µ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. 14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 µmol/kg morphine or with 14-O-MeM6SU (12 µmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED50 measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 µM) and morphine (1 µM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible.

[Parametric optimization of electroacupuncture against bone-cancer pain in rats and its intervention on mRNA expression of opioid receptor and precursor].

OBJECTIVE: To observe the intervention of electroacupuncture (EA) with different current frequencies and treatment frequencies on pain thresholt in rats with bone-cancer pain, so as to optimize treatment parameters of EA against bone cancer pain; and by measuring gene expression of opioid receptor and precursor in different tissues to preliminarily explore the possible mechanism of EA against bone cancer pain. METHODS: Ninety healthy female SD rats were randomly divided into a control group, a model group, EA groups (6 subgroups according to different frequencies) and a sham EA group, ten rats in each one. Rats in the control group were injected with 10 µL of amicrobic phosphate buffer solution (PBS) into tibial cavity; rats in the remaining groups were injected with Walker 256 cancer cells to establish model of bone-cancer pain. No treatment was given to rats in the control group and model group; rats in the EA groups were treated with EA at bilateral "Housanli" (ST 36) and "Genduan" with 3 different current frequencies (2 Hz, 100 Hz and 2 Hz/100 Hz), once a day and once every other day, 30 min per treatment (1mA for 15 min, 2 mA for 15 min); rats in the sham EA group were treated with identical acupoints as the EA group, but the acupoints were needled subcutaneously and EA was connected with power off. All the treatment was given for 14 days. Dynamic plantar aesthesiometer was applied to measure the paw withdrawal thresholds (PWTs) of the affected side before the model establishment, 6d, 8d, 10d, 12d, 14d, 16d, 18d, and 20d after model establishment. The mRNA expressions of µ-opioid receptor (MOR), κ-opioid receptor (KOR), δ-opioid receptor (DOR), proopiomelanocortin (POMC) and prodynorphin (PDYN) in dorsal root ganglion (DRG) and lumbar spinal cord dorsal horn (SCDH) of L4-L6 of the affected side were detected by PCR method. RESULTS: There were no differences in PWTs among all groups before model establishment (P>0. 05). Each time point after model establishment, PWTs in model group were obviously lower than those in the control group (all P<0. 01). Compared with the model group, PWTs in each EA subgroup were all increased (all P<0.05), but the differences at different time points were not significant among EA subgroups (P>0.05). The mRNA expressions of MOR, KOR, POMC, and PDYN in L4-L6 DRG in the 2 Hz/100 Hz II group were significantly higher than those in model group (P<0. 05, P<0. 01), while the mRNA expressions of MOR, KOR, DOR, POMC and PDYN in SCDH were not different compared with the model group (P>0. 05). CONCLUSION: EA treatment has obvious analgesic effect on bone-cancer pain, however, its effect is not related with current frequency and treating frequency. EA against bone-cancer pain may be related with increasing the mRNA expression of some peripheral opioid receptors and precursor.

Role of nociceptin/orphanin FQ in thermoregulation.

Nociceptin/Orphanin FQ (N/OFQ) is a 17-amino acid peptide that binds to the nociceptin receptor (NOP). N/OFQ and NOP receptors are expressed in numerous brain areas. The generation of specific agonists, antagonists and receptor-deficient mice or rats has enabled progress in elucidating the biological functions of N/OFQ. These tools have been employed to identify the biological significance of the N/OFQ system and how it interacts with other endogenous systems to regulate several body functions. The present review focuses on the role of N/OFQ in the regulation of body temperature and its relationship with energy balance. Critical evaluation of the literature data suggests that N/OFQ, acting through the NOP receptor, may cause hypothermia by influencing the complex thermoregulatory system that operates as a federation of independent thermoeffector loops to control body temperature at the hypothalamic level. Furthermore, N/OFQ counteracts hyperthermia elicited by cannabinoids or µ-opioid agonists. N/OFQ-induced hypothermia is prevented by ω-conotoxin GVIA, an N-type calcium channel blocker. Hypothermia induced by N/OFQ is considered within the framework of the complex action that this neuropeptide exerts on energy balance. Energy stores are regulated through the complex neural controls exerted on both food intake and energy expenditure. In laboratory rodents, N/OFQ stimulates consummatory behavior and decreases energy expenditure. Taken together, these studies support the idea that N/OFQ contributes to the regulation of energy balance by acting as an "anabolic" neuropeptide as it elicits effects similar to those produced in the hypothalamus by other neuropeptides such as orexins and neuropeptide Y.

Orally administered novel cyclic pentapeptide P-317 alleviates symptoms of diarrhoea-predominant irritable bowel syndrome.

OBJECTIVE: The aim of our study was to characterize the effect of P-317, a novel cyclic derivative of morphiceptin, on gastrointestinal (GI) motility and abdominal pain in mouse models mimicking symptoms of diarrhoea-predominant irritable bowel syndrome (IBS-D). METHODS: The effect of P-317 on mouse intestinal motility was characterized in vitro and in vivo in physiological and pathophysiological conditions. The antinociceptive action of P-317 was characterized in the mustard oil-induced abdominal pain model and the writhing test. Locomotor activity and grip-strength tests were used to evaluate the effect of P-317 in the central nervous system (CNS). To translate our study to clinical conditions, the semi-quantitative expression of µ-opioid receptors (MOP) and κ-opioid receptors (KOP) messenger RNA (mRNA) in human colonic samples from IBS-D patients was quantified. KEY FINDINGS: In vitro, P-317 (10(-10) -10(-6) M) inhibited colonic and ileal smooth muscle contractions in a concentration-dependent, ß-funaltrexamine and nor-binaltorphimine-reversible manner. In vivo, P-317 (0.1 mg/kg, i.p. and 1 mg/kg, p.o.) inhibited GI transit, displayed a potent antinociceptive action in abdominal pain tests and did not influence the CNS. CONCLUSION: P-317 produced a potent analgesic and antidiarrhoeal action in the mouse GI tract after oral administration. Given lower expression of MOP and KOP mRNA in IBS-D patients, P-317 is a promising peptide-based drug candidate for IBS-D therapy.

Activation of amygdala opioid receptors by electroacupuncture of Feng-Chi (GB20) acupoints exacerbates focal epilepsy.

BACKGROUND: The effect of seizure suppression by acupuncture of Feng-Chi (GB20) acupoints has been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot), however, there is a lack of scientific evidence to prove it. This current study was designed to elucidate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi (GB20) acupoints on the epileptic activity by employing an animal model of focal epilepsy. METHODS: Administration of pilocarpine into the left central nucleus of amygdala (CeA) induced the focal epilepsy in rats. Rats received a 30-min 100 Hz EA stimulation of bilateral Feng-Chi acupoints per day, beginning at 30 minutes before the dark period and performing in three consecutive days. The broad-spectrum opioid receptor antagonist (naloxone), µ-receptor antagonist (naloxonazine), δ-receptor antagonist (naltrindole) and κ-receptor antagonist (nor-binaltorphimine) were administered directly into the CeA to elucidate the involvement of CeA opioid receptors in the EA effect. RESULTS: High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints did not suppress the pilocarpine-induced epileptiform electroencephalograms (EEGs), whereas it further increased the duration of epileptiform EEGs. We also observed that epilepsy occurred while 100 Hz EA stimulation of Feng-Chi acupoints was delivered into naïve rats. EA-induced augmentation of epileptic activity was blocked by microinjection of naloxone, µ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting that activation of opioid receptors in the CeA mediates EA-exacerbated epilepsy. CONCLUSIONS: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints has no effect to protect against pilocarpine-induced focal epilepsy; in contrast, EA further exacerbated focal epilepsy induced by pilocarpine. Opioid receptors in the CeA mediated EA-induced exacerbation of focal epilepsy.

Comparison of the transcriptional responses induced by acute morphine, methadone and buprenorphine.

Despite their widespread use in opioid maintenance treatment and pain management, little is known about the intracellular effectors of methadone and buprenorphine and the transcriptional responses they induce. We therefore studied the acute effects of these two opioids in rats, comparing our observations with those for the reference molecule, morphine. We determined the analgesic ED50 of the three molecules in the tail flick test, to ensure that transcriptional effects were compared between doses of equivalent analgesic effect. We analysed changes in gene expression over time in three cerebral structures involved in several opioid behaviours-the dorsal striatum, thalamus and nucleus accumbens-by real-time quantitative PCR. We analysed the expression of genes encoding proteins of the endogenous opioid system in parallel with that of Fos, a marker of neuronal activation. The acute transcriptional effects of methadone resembled those of morphine more closely than did those of buprenorphine, in terms of kinetics and intensities. Our results provide the first evidence that these two drugs widely used in pain management and opioid maintenance treatment can disturb the regulation of endogenous opioid system genes and induce molecular outcomes different from those observed with morphine.

Caloric restriction increases the sensitivity to the hyperphagic effect of nociceptin/orphanin FQ limiting its ability to reduce binge eating in female rats.

RATIONALE: Nociceptin/orphanin FQ (N/OFQ) is a functional antagonist of corticotrophin-releasing factor, the main mediator of the stress response. Stress represents a key determinant of binge eating (BE) for highly palatable food (HPF). OBJECTIVES: In relation to the antistress properties of N/OFQ, we evaluated its effect on BE. After the observation that episodes of food restriction increase the sensitivity to its hyperphagic effects, the function of NOP receptor and N/OFQ was investigated after cycles of food restrictions. MATERIALS AND METHODS: In BE experiments, four groups were used: rats fed normally and not stressed or stressed, rats exposed to cycles of restriction/refeeding and then stressed, or not stressed. In the other experiments, two groups were used: rats exposed or not to food restriction. RESULTS: Only restricted and stressed rats exhibited BE for HPF (containing chocolate cream). Intracerebroventricular injections of N/OFQ of 0.5 nmol/rat significantly reduced BE. N/OFQ 1 nmol/rat did not reduce BE but significantly increased HPF intake following food restrictions. Cycles of food restriction increased animals' sensitivity to the hyperphagic effect of N/OFQ for HPF. In situ hybridization studies following food restrictions showed decreased ppN/OFQ mRNA expression in the bed nucleus of the stria terminalis and increased expression of ppN/OFQ and NOP receptor mRNA in the ventral tegmental area and in the ventromedial hypothalamus, respectively. CONCLUSIONS: These findings indicate that N/OFQ slightly reduces BE at low doses, while higher doses increase HPF intake, due to increased sensitivity to its hyperphagic effect following a history of caloric restrictions.

[Effects of electroacupuncture preconditioning at "Neiguan" (PC 6) on gene expression of myocardial opioid receptors in rats with myocardial ischemia-reperfusion injury].

OBJECTIVE: To explore the protective effects of electroacupuncture preconditioning at "Neiguan" (PC 6) on myocardial ischemia reperfusion injury (MIRI) and the mechanism. METHODS: Forty-eight male Wistar rats were randomly divided into a sham operation group (group N), a MIRI group (group M) and an electroacupuncture group (group E). The MIRI model was generated by ligating the left anterior descending artery (LAD) for 30 min followed by reperfusion for 120 min. The partition sutures in group N were passed under the LAD without ligation. The rats in group E were pretreated with electroacupuncture applied at bilateral "Neiguan" (PC 6) for 20 min once a day on 3 consecutive days before ischemia. The area at risk and infarct size and the serum levels of CK-MB and LDH were measured. The morphological changes were observed by light microscopy. The mRNA expressions of delta and kappa opioid receptor (DOR and KOR) were determined by real-time RT-PCR. RESULTS: The myocardial infarct size in group E was significant smaller than that in group M (P < 0.05). The contents of CK-MB and LDH were (980 +/- 92) U/L and (2 743 +/- 124) U/L in group M which were significant higher than (312 +/- 41) U/L and (530 +/- 56) U/L in group N (both P < 0.01) and (572 +/- 70) U/L and (1 819 +/- 97) U/L in group E (both P < 0.01). The mRNA expressions of DOR and KOR had no significant difference between group M and N (both P > 0.05), while the DOR expression in group E was significant higher than that in group M and N (both P < 0.01). There was no significant difference in KOR expression among three groups (all P > 0.05). CONCLUSION: The cardiac protective effects of electroacupuncture preconditioning at "Neiguan" (PC 6) on MIRI may be mediated by increasing expression of delta opioid receptor in myocardium.

Effect of tooth pulp and periaqueductal central gray stimulation on the expression of genes encoding the selected neuropeptides and opioid receptors in the mesencephalon, hypothalamus and thalamus in rats.

Nociceptive stimulation has been considered to affect the expression of genes encoding endogenous neuropeptides and their receptors. The effect of electric stimulation of the tooth pulp and/or periaqueductal gray (PAG) in rats on mRNA levels of the selected neuropeptides and opioid receptors (ORs) was investigated in comparison with control group, without stimulation. The levels of mRNA for the selected neuropeptides: galanin (GAL), vasopressin (AVP), oxytocin (OT), substance P (SP), somatostatin (SOM), vasoactive intestinal peptide (VIP), endomorphin-2 (EM-2), and opioid receptors: MOR, DOR and KOR in mesencephalic, hypothalamic and thalamic tissues were determined by real-time PCR. It was demonstrated that in the control group expression of the tested neuropeptides was at a very low level in the mesencephalon and thalamus, but at the higher level in the hypothalamus. The highest expression of ORs was observed in the mesencephalon. Nociceptive tooth pulp stimulation had the strongest effect in the hypothalamus, elevating mRNA levels of all tested neuropeptides except SOM. Electric stimulation of PAG either did not change or down-regulated mRNA levels of the neuropeptides in the cerebral structures. Simultaneous stimulation of PAG and tooth pulp either did not affect mRNA levels of the investigated neuropeptides or caused their slight decrease versus tooth pulp stimulation. The noxious stimulation of tooth pulp increased also the levels of OR mRNAs, while stimulation of PAG had the opposite effect. The above results demonstrated that tooth pulp stimulation significantly up-regulated the mRNA levels for a number of neuropeptides and all three types of ORs in the rat brain, which would result in more potent antinociception. In contrast, PAG stimulation down-regulated the mRNA levels of several neuropeptides and ORs in the cerebral tissues, which would cause decreased synthesis of ORs. The obtained results represent a new insight into the mechanism of orofacial pain.