Electroacupuncture alleviates the relapse of pain-related aversive memory by activating KOR and inhibiting GABAergic neurons in the insular cortex.

Pain-related aversive memory is common in chronic pain patients. Electroacupuncture has been demonstrated to block pain-related aversive memory. The insular cortex is a key region closely related to aversive behaviors. In our study, a potential mechanism underlying the effect of electroacupuncture treatment on pain-related aversive memory behaviors relative to the insular cortex was investigated. Our study used the chemogenetic method, pharmacological method, electroacupuncture intervention, and behavioral detection. Our study showed that both inhibition of gamma-aminobutyric acidergic neurons and activation of the kappa opioid receptor in the insular cortex blocked the pain-related aversive memory behaviors induced by 2 crossover injections of carrageenan in mice; conversely, both the activation of gamma-aminobutyric acidergic neurons and inhibition of kappa opioid receptor in the insular cortex play similar roles in inducing pain-related aversive memory behaviors following 2 crossover injections of carrageenan. In addition, activation of gamma-aminobutyric acidergic neurons in the insular cortex reversed the effect of kappa opioid receptor activation in the insular cortex. Moreover, electroacupuncture effectively blocked pain-related aversive memory behaviors in model mice, which was reversed by both activation of gamma-aminobutyric acidergic neurons and inhibition of kappa opioid receptor in the insular cortex. The effect of electroacupuncture on blocking pain-related aversive memory behaviors may be related to the activation of the kappa opioid receptor and inhibition of gamma-aminobutyric acidergic neurons in the insular cortex.

Spatial quantification of single cell mRNA and ligand binding of the kappa opioid receptor in the rat hypothalamus.

Detailed quantification of brain tissue provides a deeper understanding of changes in expression and function. We have created a pipeline to study the detailed expression patterns of the kappa opioid receptor in the rat hypothalamus using high resolution fluorescence microscopy and receptor autoradiography. The workflow involved structured serial sampling of rat hypothalamic nuclei, in situ detection of mRNA and receptor expression, and advanced image analysis. Our results demonstrate how maintaining spatial information can lead to increased understanding of RNA and protein expression. In addition, we show the detailed expression patterns of the kappa opioid receptor in the rat hypothalamus.

Design of a Stable Cyclic Peptide Analgesic Derived from Sunflower Seeds that Targets the κ-Opioid Receptor for the Treatment of Chronic Abdominal Pain.

The rising opioid crisis has become a worldwide societal and public health burden, resulting from the abuse of prescription opioids. Targeting the κ-opioid receptor (KOR) in the periphery has emerged as a powerful approach to develop novel pain medications without central side effects. Inspired by the traditional use of sunflower (Helianthus annuus) preparations for analgesic purposes, we developed novel stabilized KOR ligands (termed as helianorphins) by incorporating different dynorphin A sequence fragments into a cyclic sunflower peptide scaffold. As a result, helianorphin-19 selectively bound to and fully activated the KOR with nanomolar potency. Importantly, helianorphin-19 exhibited strong KOR-specific peripheral analgesic activity in a mouse model of chronic visceral pain, without inducing unwanted central effects on motor coordination/sedation. Our study provides a proof of principle that cyclic peptides from plants may be used as templates to develop potent and stable peptide analgesics applicable via enteric administration by targeting the peripheral KOR for the treatment of chronic abdominal pain.

Effects of processed Aconiti tuber on the extinction and reinstatement of morphine-induced conditioned place preference in rats.

AIM OF THE STUDY: To investigate the effect of processed Aconiti tuber (PAT) administered during or after the time of conditioned place preference (CPP) training on the extinction and reinstatement of morphine-priming CPP in rats. The dynorphin level in rats' nucleus accumbens (NAc) is detected as a target of the Dynorphin/Kappa Opioid Receptor (KOR) system for the possible mechanism. MATERIALS AND METHODS: Eight groups of rats were subcutaneously (s.c.) injected with morphine (10mg/kg) (on days 2,4,6,8) or saline (1ml/kg) (on days 3,5,7,9) alternately for 8 days. Five groups, including groups (Mor + Water, Mor + PAT (1.0/3.0g/kg) (S) and Sal + PAT(1.0/3.0g/kg)), were orally given distilled water or PAT 1.0 or 3.0 g/kg daily on days 1-8 during CPP training while other three groups, including groups (Sal + Water and Mor + PAT (1.0/3.0g/kg)(P), were given distilled water or PAT daily from day 10 until CPP was extinct. Morphine 1mg/kg (s.c.) was used to reinstate the extinct CPP and the CPP scores were recorded. The dynorphin concentration in nucleus accumbens (NAc) was assayed by radioimmunoassay after the last CPP measurement. RESULTS: 1) The CPP extinction shortened in Mor + PAT (1.0/3.0 g/kg) (S) groups but extended in Mor + PAT (1.0/3.0 g/kg)(P) groups. 2) Morphine-priming CPP did not change either in Mor + PAT (1.0/3.0 g/kg) (S) or Mor + PAT (1.0/3.0 g/kg)(P) groups. 3) The dynorphin concentration in NAc increased either in Mor + PAT (1.0/3.0 g/kg)(S) or Mor + PAT (1.0/3.0 g/kg)(P) groups. CONCLUSIONS: 1) PAT shortened the extinction from morphine induced CPP when administrated before CPP acquisition, whereas it extended the extinction when administrated after CPP formation. 2) PAT administrated during or after CPP training did not affect morphine-priming reinstatement of morphine induced CPP. 3) Dynorphin/KOR system might be a target to regulate morphine-induced CPP extinction but not reinstatement.

Toosendanin-induced apoptosis in colorectal cancer cells is associated with the κ-opioid receptor/ß-catenin signaling axis.

Developing new drugs for killing colorectal cancer (CRC) cells is urgently needed. Here, we explored the antitumor effects of toosendanin (TSN) in CRC, as well as explored its antitumor mechanisms and direct targets. Cell proliferation and apoptosis were analyzed by CCK8, colony formation, real-time cell impedance and flow cytometry. The signaling pathway and Wnt activity were analyzed by Wnt luciferase activity assay, quantitative real-time PCR and western blot. The interaction between TSN and the κ-opioid receptor was analyzed by a molecular docking simulation. BALB/c nude mice were used to detect the effects of TSN on tumor growth in vivo. We found that TSN inhibited proliferation, induced G1 phase arrest and caused caspase-dependent apoptosis in both 5-FU-sensitive and 5-FU-resistant CRC cells. Moreover, TSN effectively inhibited CRC growth in vivo. In terms of the mechanism, TSN inhibited Wnt/ß-catenin signaling in CRC cells, and the molecular docking results showed that TSN could bind to κ-opioid receptors directly. Additionally, TSN-induced apoptosis and ß-catenin decline were both reversed by the selective κ-opioid receptor agonist U50,488H. Our data demonstrate that TSN-induced apoptosis in CRC cells is associated with the κ-opioid receptor/ß-catenin signaling axis, and TSN has promising potential as an antitumor agent for CRC treatment.

Behavioral effects of the kappa opioid receptor partial agonist nalmefene in tests relevant to depression.

Compounds with high affinity at kappa and mu opioid receptors may have clinical utility in treating major depressive disorder. Nalmefene (NMF) is a partial kappa opioid receptor agonist and potent mu opioid receptor antagonist, but there has been no preclinical evaluation of NMF in rodent tests relevant to depression and anxiety. To address this, the effects of NMF on neurochemical and behavioral endpoints in C57BL/6J mice were examined and contrasted with a structurally related analog, naltrexone (NTX). NMF exhibited kappa opioid receptor agonist activity, measured as a reduction in extracellular dopamine release in the nucleus accumbens using in vivo microdialysis following acute but not chronic administration. In the mouse forced swim test, female mice were more responsive to higher doses of NMF and NTX compared to male mice. The behavioral effects of NMF in the forced swim test were blocked in Oprk1-/- and Oprm1-/- mice. Conversely, the effects of NTX were blocked only in Oprm1-/- mice. These results indicate that both kappa and mu opioid receptors mediate the behavioral effects of NMF, but the effects of NTX in this test were modified only by mu opioid receptor engagement. Unlike NTX, NMF did not produce conditioned place aversion in either sex. Finally, NMF's activity in the marble burying test and forced swim test were retained following chronic administration. The sustained effects exerted by NMF on tests that are sensitive to antidepressant and anxiolytic compounds support further investigation of NMF as a potential therapeutic for depression.

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.

Central nicotine induces browning through hypothalamic κ opioid receptor.

Increased body weight is a major factor that interferes with smoking cessation. Nicotine, the main bioactive compound in tobacco, has been demonstrated to have an impact on energy balance, since it affects both feeding and energy expenditure at the central level. Among the central actions of nicotine on body weight, much attention has been focused on its effect on brown adipose tissue (BAT) thermogenesis, though its effect on browning of white adipose tissue (WAT) is unclear. Here, we show that nicotine induces the browning of WAT through a central mechanism and that this effect is dependent on the κ opioid receptor (KOR), specifically in the lateral hypothalamic area (LHA). Consistent with these findings, smokers show higher levels of uncoupling protein 1 (UCP1) expression in WAT, which correlates with smoking status. These data demonstrate that central nicotine-induced modulation of WAT browning may be a target against human obesity.

Alterations in the Activity of Spinal and Thalamic Opioid Systems in a Mice Neuropathic Pain Model.

Clinical studies have reported lower effectivity of opioid drugs in therapy of neuropathic pain. Therefore, to determine the changes in endogenous opioid systems in this pain more precisely, we have studied the changes in the pain-related behavior on days 1, 14, and 28 following a chronic constriction injury (CCI) to the sciatic nerve in mice. In parallel, we have studied the changes of µ-(MOP), δ-(DOP) and κ-(KOP) receptors, proenkephalin (PENK) and prodynorphin (PDYN) mRNA levels, as well as GTPγS binding of opioid receptors on the ipsi- and contralateral parts of the spinal cord and thalamus on the 14th day following CCI, as on this day the greatest manifestation of pain-related behavior was observed. On ipsilateral spinal cord, the decrease in MOP/DOP/KOP receptor and increase in PDYN/PENK mRNA expression was observed. In thalamus, MOP/DOP/KOP receptor expression decreased contralaterally. On ipsilateral side, there were no changes in PDYN/PENK or DOP/KOP receptor expression, but MOP mRNA decreased. The spinal GTPγS binding of MOP/DOP/KOP receptor ligands decreased on the ipsilateral side, yet the effect was less pronounced for DOP receptor ligands. In thalamus, a decrease was observed on the contralateral side for all opioid receptor ligands, especially for DOP ligand. A less pronounced decrease in GTPγS binding of spinal DOP ligands may indicate a weaker stimulation of ascending nociceptive pathways, which could explain the absence of decreased activity of DOP receptor ligands in neuropathy. These findings may suggest that drugs with a higher affinity for the DOP receptor will perform better in neuropathic pain.

Evidence That Dynorphin Acts Upon KNDy and GnRH Neurons During GnRH Pulse Termination in the Ewe.

A subpopulation of neurons located within the arcuate nucleus, colocalizing kisspeptin, neurokinin B, and dynorphin (Dyn; termed KNDy neurons), represents key mediators of pulsatile GnRH secretion. The KNDy model of GnRH pulse generation proposes that Dyn terminates each pulse. However, it is unknown where and when during a pulse that Dyn is released to inhibit GnRH secretion. Dyn acts via the κ opioid receptor (KOR), and KOR is present in KNDy and GnRH neurons in sheep. KOR, similar to other G protein-coupled receptors, are internalized after exposure to ligand, and thus internalization can be used as a marker of endogenous Dyn release. Thus, we hypothesized that KOR will be internalized at pulse termination in both KNDy and GnRH neurons. To test this hypothesis, GnRH pulses were induced in gonad-intact anestrous ewes by injection of neurokinin B (NKB) into the third ventricle and animals were euthanized at times of either pulse onset or termination. NKB injections produced increased internalization of KOR within KNDy neurons during both pulse onset and termination. In contrast, KOR internalization into GnRH neurons was seen only during pulse termination, and only in GnRH neurons within the mediobasal hypothalamus (MBH). Overall, our results indicate that Dyn is released onto KNDy cells at the time of pulse onset, and continues to be released during the duration of the pulse. In contrast, Dyn is released onto MBH GnRH neurons only at pulse termination and thus actions of Dyn upon KNDy and GnRH cell bodies may be critical for pulse termination.

Structurally Related Kappa Opioid Receptor Agonists with Substantial Differential Signaling Bias: Neuroendocrine and Behavioral Effects in C57BL6 Mice.

Background: The kappa opioid receptor system has been revealed as a potential pharmacotherapeutic target for the treatment of addictions to substances of abuse. Kappa opioid receptor agonists have been shown to block the rewarding and dopamine-releasing effects of psychostimulants. Recent investigations have profiled the in vivo effects of compounds biased towards G-protein-mediated signaling, with less potent arrestin-mediated signaling. The compounds studied here derive from a series of trialkylamines: N-substituted-N- phenylethyl-N-3-hydroxyphenylethyl-amine, with N-substituents including n-butyl (BPHA), methylcyclobutyl (MCBPHA), and methylcyclopentyl (MCPPHA). Methods: BPHA, MCBPHA, and MCPPHA were characterized in vitro in a kappa opioid receptor-expressing cell line in binding assays and functional assays. We also tested the compounds in C57BL6 mice, assaying incoordination with rotarod, as well as circulating levels of the neuroendocrine kappa opioid receptor biomarker, prolactin. Results: BPHA, MCBPHA, and MCPPHA showed full kappa opioid receptor agonism for G-protein coupling compared with the reference compound U69,593. BPHA showed no measurable ß-arrestin-2 recruitment, indicating that it is extremely G-protein biased. MCBPHA and MCPPHA, however, showed submaximal efficacy for recruiting ß-arrestin-2. Studies in C57BL6 mice reveal that all compounds stimulate release of prolactin, consistent with dependence on G-protein signaling. MCBPHA and MCPPHA result in rotarod incoordination, whereas BPHA does not, consistent with the reported requirement of intact kappa opioid receptor/ß-arrestin-2 mediated coupling for kappa opioid receptor agonist-induced rotarod incoordination. Conclusions: BPHA, MCBPHA, and MCPPHA are thus novel differentially G-protein-biased kappa opioid receptor agonists. They can be used to investigate how signaling pathways mediate kappa opioid receptor effects in vitro and in vivo and to explore the effects of candidate kappa opioid receptor-targeted pharmacotherapeutics.

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2.

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH2 (C-36) and Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2 (F-81). The ability of these peptides to cross the blood-brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.

[New thoughts in mechanism research of acupuncture for myocardial stunning from κ-opioid receptor signaling pathway].

By reviewing the literature regarding the development mechanism of myocardial stunning, effects of acupuncture on myocardial ischemic injury, and correlation between acupuncture and κ-opioid receptor, it was suggested that acupuncture was highly likely to act on κ-opioid receptor in myocardial cells, and directly treated myocardial malfunction induced by myocardial stunning through κ-opioid receptor and its signaling pathway. In addition, acupuncture could inhabit the signaling pathway of adrenoceptor ß1, one of the main functional receptors, to indirectly improve myocardial ischemic injury. From κ-opioid receptor signaling pathway, the action mechanism of acupuncture for prevention and treatment of myocardial stunning was discussed in this paper, hoping to provide new ideas for possible mechanism of acupuncture for myocardial ischemic injury.

Epigenetic regulation of the kappa opioid receptor gene by an insertion-deletion in the promoter region.

Preclinical and clinical studies have demonstrated that the kappa opioid receptor (KOR) regulates reward, hedonic tone and emotions. At therapeutic level, on-going clinical trials are assessing the potential of targeting the KOR for the management of depression, anxiety disorders and substance use disorders. However, genetic polymorphisms in the KOR gene that potentially contribute to its implication in these phenotypes have been poorly studied. Here we investigated an insertion-deletion in the promoter region of KOR (rs35566036), recently associated with alcohol addiction, in a cohort of depressed subjects who died by suicide, as well as psychiatrically healthy individuals. Focusing on 3 brain regions (anterior insula, anterior cingulate cortex, and mediodorsal thalamus), we characterized the functional impact of this structural variant on the expression and patterns of DNA methylation of the KOR gene, using qPCR and targeted Bisulfite-Sequencing, respectively. While there was no significant change in the expression of KOR as a function of the insertion-deletion, or as a function of disease status in any brain region, we found that this variant strongly determines DNA methylation in KOR promoter, leading to a significant decrease in methylation levels of 8 nearby CpG dinucleotides located approximately 500 base pairs upstream the transcription start site. In addition, our results suggest a possible association between the insertion-deletion and depression; however, this result should be tested in larger populations. In sum, in this study we uncovered an epigenetic mechanism potentially contributing to KOR dysfunction in carriers of the insertion-deletion.

Lateralized kappa opioid receptor signaling from the amygdala central nucleus promotes stress-induced functional pain.

The response of diffuse noxious inhibitory controls (DNIC) is often decreased, or lost, in stress-related functional pain syndromes. Because the dynorphin/kappa opioid receptor (KOR) pathway is activated by stress, we determined its role in DNIC using a model of stress-induced functional pain. Male, Sprague-Dawley rats were primed for 7 days with systemic morphine resulting in opioid-induced hyperalgesia. Fourteen days after priming, when hyperalgesia was resolved, rats were exposed to environmental stress and DNIC was evaluated by measuring hind paw response threshold to noxious pressure (test stimulus) after capsaicin injection in the forepaw (conditioning stimulus). Morphine priming without stress did not alter DNIC. However, stress produced a loss of DNIC in morphine-primed rats in both hind paws that was abolished by systemic administration of the KOR antagonist, nor-binaltorphimine (nor-BNI). Microinjection of nor-BNI into the right, but not left, central nucleus of the amygdala (CeA) prevented the loss of DNIC in morphine-primed rats. Diffuse noxious inhibitory controls were not modulated by bilateral nor-BNI in the rostral ventromedial medulla. Stress increased dynorphin content in both the left and right CeA of primed rats, reaching significance only in the right CeA; no change was observed in the rostral ventromedial medulla or hypothalamus. Although morphine priming alone is not sufficient to influence DNIC, it establishes a state of latent sensitization that amplifies the consequences of stress. After priming, stress-induced dynorphin/KOR signaling from the right CeA inhibits DNIC in both hind paws, likely reflecting enhanced descending facilitation that masks descending inhibition. Kappa opioid receptor antagonists may provide a new therapeutic strategy for stress-related functional pain disorders.

New tetranorlabdanoic acid from aerial parts of Salvia aethiopis.

Salvia aethiopis is a perennial plant native to Eurasia and known by the common names Mediterranean sage or African sage. This plant has been used in Iranian medicine as a carminative and tonic. The ethanolic extract of aerial part of S. aethiopis exhibited moderate to weak activity towards delta and kappa opioid receptors (46.3 and 45.3% displacement, respectively). Chromatographic purification of the ethanolic extract on silica gel column led to isolation of one new: 3α-hydroxy-8α-acetoxy-13,14,15,16-tetranorlabdan-12-oic acid (I) and three known compounds: sesquiterpene spathulenol (II), ß-sitosterol (III) and ß-sitosterol-3-O-glucoside (IV). The structures of all isolated compounds were elucidated by their NMR (1D and 2D) and MS spectral data. All the fractions and isolated compounds were tested for cannabinoid and opioid receptor binding assays.

Concurrent bullatine A enhances morphine antinociception and inhibits morphine antinociceptive tolerance by indirect activation of spinal κ-opioid receptors.

ETHNOPHARMACOLOGICAL RELEVANCE: Bullatine A, a C20-diterpenoid alkaloid and one of the major effective ingredients in Aconiti brachypodi Radix (Xue-shang-yi-zhi-hao), can block pain hypersensitivity in a variety of rodent models through expression of spinal microglial dynorphin A. AIM OF THE STUDY: To assess the interaction between bullatine A and morphine on antinociception in acute nociception and pain hypersensitivity states, with the exogenous synthetic dynorphin A as a comparison MATERIALS AND METHODS: Spinal nerve ligation-induced neuropathic rats and naïve mice were used for assessing the acute and chronic interactions of bullatine A/dynorphin A with morphine. RESULTS: Single subcutaneous injection of bullatine A or dynorphin A(1-17) did not either alter formalin- and thermally (hot-plate and water immersion tests)-induced acute nociception or potentiate morphine antinociception in naïve mice. In contrast, bullatine A dose-dependently inhibited formalin-induced tonic pain with the efficacy of 54% inhibition and the half-effective dose of 0.9mg/kg. Concurrent bullatine A additively enhanced morphine antinociception. In neuropathic rats, the antinociceptive effects of multiple bidaily intrathecal injections of bullatine A and dynorphin A remained consistent over 13 days, whereas morphine produced progressive and complete tolerance to antinociception, which was completely inhibited by concurrent bullatine A and dynorphin A. A single intrathecal injection of bullatine A and dynorphin A immediately reversed established morphine tolerance in neuropathic rats, although the blockade was a less degree in the thermally induced mouse acute nociceptive tests. The inhibitory effects of bullatine A and dynorphin A on morphine tolerance were immediately and completely attenuated by intrathecal dynorphin A antibody and/or selective κ-opioid receptor antagonist GNTI. CONCLUSION: These results suggest that bullatine A produces antinociception without induction of tolerance and inhibits morphine antinociceptive tolerance, and provide pharmacological basis for concurrent bullatine A and morphine treatment for chronic pain and morphine analgesic tolerance.

κ-Opioid Receptor Is Colocalized in GnRH and KNDy Cells in the Female Ovine and Rat Brain.

Kisspeptin-neurokinin B-dynorphin (KNDy) cells of the hypothalamus are a key component in the neuroendocrine regulation of GnRH secretion. Evidence in sheep and other species suggests that dynorphin released by KNDy cells inhibits pulsatile GnRH secretion by acting upon κ-opioid receptors (KOR). However, the precise anatomical location and neurochemical phenotype of KOR-expressing cells in sheep remain unknown. To this end, we determined the distribution of KOR mRNA and protein in the brains of luteal phase ewes, using an ovine specific KOR mRNA probe for in situ hybridization and an antibody whose specificity we confirmed by Western blot analyses and blocking peptide controls. KOR cells were observed in a number of regions, including the preoptic area (POA); anterior hypothalamic area; supraoptic and paraventricular nuclei; ventromedial, dorsomedial, and lateral hypothalamus; and arcuate nucleus. Next, we determined whether KOR is colocalized in KNDy and/or GnRH cells. Dual-label immunofluorescence and confocal analysis of the KNDy population showed a high degree of colocalization, with greater than 90% of these neurons containing KOR. Surprisingly, GnRH cells also showed high levels of colocalization in sheep, ranging from 74.4% to 95.4% for GnRH cells in the POA and medial basal hypothalamus, respectively. Similarly, 97.4% of GnRH neurons in the POA of ovariectomized, steroid-primed female rats also contained immunoreactive KOR protein. These findings suggest that the inhibitory effects of dynorphin on pulsatile GnRH secretion may occur either indirectly by actions upon KOR within the KNDy population and/or directly via the activation of KOR on GnRH cells.

Electroacupuncture relieves labour pain and influences the spinal dynorphin/κ-opioid receptor system in rats.

BACKGROUND: The dynorphin (DYN)/κ-opioid receptor (KOR) system plays a key role in the control of labour pain. Our previous clinical study reported that electroacupuncture (EA) provided intrapartum analgesia, but the underlying mechanisms of action have not been fully elucidated. AIMS: To observe the effect of EA on labour pain and to explore the underlying mechanisms of action in a rat model. METHODS: Copulation-confirmed pregnant rats (n=120) were given castor oil to induce labour. Rats remained untreated (control group, n=20) or received either meperidine (an opioid that is commonly used to treat labour pain, n=20) or EA at SP6, LI4, SP6+LI4 or SP10 (four groups, n=20 each). Labour pain was evaluated by the warm water tail-flick test. Serum DYN values were measured by ELISA. Protein and mRNA expression of prodynorphin (PDYN, the precursor protein of DYN) and KOR were analysed by Western blotting and real-time PCR, respectively. RESULTS: EA treatment at all acupuncture point combinations studied significantly relieved labour pain and increased serum DYN concentrations, to a degree similar to that achieved with meperidine. EA notably enhanced protein expression of KOR and PDYN and mRNA expression in the lumbar spinal cord but not in the cerebral cortex. The size of effect varied by EA group in the order: SP6>LI4>SP6+LI4>SP10 for all parameters measured, indicating differential effects relating to acupuncture point selection/combination. CONCLUSIONS: The present study indicates that EA relieves labour pain, at least in part, by regulation of the spinal DYN/KOR system in a rat model.

Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of the 3- and 4-methyl substituents.

The design and discovery of JDTic as a potent and selective kappa opioid receptor antagonist used the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine pharmacophore as the lead structure. In order to determine if the 3-methyl or 4-methyl groups were necessary in JDTic and JDTic analogs for antagonistic activity, compounds 4a-c, and 4d-f which have either the 3-methyl or both the 3- and 4-methyl groups removed, respectively, from JDTic and analogs were synthesized and evaluated for their in vitro opioid receptor antagonist activities using a [(35)S]GTPγS binding assay. Other ADME properties were also assessed for selected compounds. These studies demonstrated that neither the 3-methyl or 3,4-dimethyl groups present in JDTic and analogs are required to produce potent and selective κ opioid receptor antagonists.