Opioid-receptor gene expression and localization in cancer cells

This study examined the presence and cellular localization of three types of opioid receptors (MOR, DOR and KOR) in five human cancer cell lines: MCF-7, MDA-MB-231, HT-29, MGH-U1 and SH-SY5Y. Expression levels of opioid receptors were measured quantitatively using real-time PCR, and the localizations of the receptors in the cells were determined by immunocytochemistry. All three types of opioid receptors were present in each of the five cell lines examined. However, three of the cell lines (MCF-7, HT-29 and SH-SY5Y) showed significantly higher levels of MOR mRNA and protein than the other two types of receptors (DOR and KOR). Immunocytochemistry revealed that MOR, DOR and KOR receptors were predominantly present on the surface of cell membranes, although these receptors were also occasionally present in the cell cytoplasm.     

Role of Opioid Receptors Signaling in Remote Electrostimulation - Induced Protection against Ischemia/Reperfusion Injury in Rat Hearts

Aims

Our previous studies demonstrated that remote electro-stimulation (RES) increased myocardial GSK3 phosphorylation and attenuated ischemia/ reperfusion (I/R) injury in rat hearts. However, the role of various opioid receptors (OR) subtypes in preconditioned RES-induced myocardial protection remains unknown. We investigated the role of OR subtype signaling in RES-induced cardioprotection against I/R injury of the rat heart.

Methods & Results

Male Spraque-Dawley rats were used. RES was performed on median nerves area with/without pretreatment with various receptors antagonists such as opioid receptor (OR) subtype receptors (KOR, DOR, and MOR). The expressions of Akt, GSK3, and PKCε expression were analyzed by Western blotting. When RES was preconditioned before the I/R model, the rat''s hemodynamic index, infarction size, mortality and serum CK-MB were evaluated. Our results showed that Akt, GSK3 and PKCε expression levels were significantly increased in the RES group compared to the sham group, which were blocked by pretreatment with specific antagonists targeting KOR and DOR, but not MOR subtype. Using the I/R model, the duration of arrhythmia and infarct size were both significantly attenuated in RES group. The mortality rates of the sham RES group, the RES group, RES group + KOR antagonist, RES group + DOR/MOR antagonists (KOR left), RES group + DOR antagonist, and RES group + KOR/MOR antagonists (DOR left) were 50%, 20%, 67%, 13%, 50% and 55%, respectively.

Conclusion

The mechanism of RES-induced myocardial protection against I/R injury seems to involve multiple target pathways such as Akt, KOR and/or DOR signaling.     

Co-expressions of different opioid receptor types differentially modulate their signaling via G(16)

Combinations of two different types of opioid receptors - delta-, kappa-, mu-opioid receptors (DOR, KOR, and MOR) and opioid receptor-like receptor 1 (ORL(1)) - were co-expressed with the alpha subunit of G(16) in COS-7 cells, and the ability of various selective agonists to induce activation of phospholipase Cbeta was examined. Nociceptin/orphanin FQ-induced response was enhanced when ORL(1) was co-expressed with MOR or KOR but not DOR. The kappa-agonist U50,488H induced a modest inositol phosphate formation when KOR was expressed alone or with MOR, but the response was attenuated when co-expressing with either DOR or ORL(1). It is suggested that the co-expressions of two different opioid receptor types indeed modify their downstream signaling events.     

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu,delta, and kappa opioid receptors

In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki?=?0.27, 0.46, and 0.87?nM; EC₅₀?=?3.47, 1.45, and 13.5?nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.     

Detection of kappa and delta opioid receptors in skin--outside the nervous system

Opioid receptors (OR) are widely expressed in the central nervous system (CNS). Opioid antinociception might be initiated by activation of OR outside the CNS, indicating targeting of peripheral OR could be useful in the treatment of chronic pain. This study was designed to detect OR in skin tissues of healthy volunteers at both mRNA and protein levels. Skin samples from 10 healthy individuals were investigated. Total isolated RNAs were reverse transcribed, amplified and quantified by real-time PCR. Tissue and skin fibroblast OR protein was detected by immunohistochemistry, Western blot, and immunofluorescence. All skin tissue samples expressed delta- (DOR) and kappa-OR (KOR) mRNAs. Using immunohistochemistry, DOR and KOR were localized in skin fibroblast-like and mononuclear cells. Skin fibroblasts in culture expressed DOR and KOR mRNA. Using immunofluorescence, both DOR and KOR proteins were expressed predominantly on the cell membrane with minor staining in the cytoplasm. We suggest that enhanced expression of DOR and KOR in skin justifies the exploration of selective novel delta and kappa agonists for local pain treatment.     

mu-Opioid receptors desensitize less rapidly than delta-opioid receptors due to less efficient activation of arrestin

Receptor desensitization by G-protein receptor kinases (GRK) and arrestins is likely to be an important component underlying the development of tolerance to opioid drugs. Reconstitution of this process in Xenopus oocytes revealed distinct differences in the kinetics of GRK and arrestin regulation of the closely related opioid receptors mu (MOR), delta (DOR), and kappa (KOR). We demonstrated that under identical conditions, GRK and arrestin-dependent desensitization of MOR proceeds dramatically slower than that of DOR. Furthermore, GRK3 phosphorylation sites required for opioid receptor desensitization also greatly differ. The determinants for DOR and KOR desensitization reside in the carboxyl-terminal tail, whereas MOR depends on Thr-180 in the second intracellular loop. Although this later finding might indicate an inefficient phosphorylation of MOR Thr-180, increasing the amount of arrestin expressed greatly increased the rate of MOR desensitization to a rate comparable with that of DOR. Similarly, coexpression of a constitutively active arrestin 2(R169E) with MOR and DOR desensitized both receptors in an agonist-dependent, GRK-independent manner at rates that were indistinguishable. Together, these data suggest that it is the activation of arrestin, rather than its binding, that is the rate-limiting step in MOR desensitization. In addition, mutation of Thr-161 in DOR, homologous to MOR Thr-180, significantly inhibited the faster desensitization of DOR. These results suggest that DOR desensitization involves phosphorylation of both the carboxyl-terminal tail and the second intracellular loop that together leads to a more efficient activation of arrestin and thus faster desensitization.     

Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain

The analgesic effect of delta-opioid receptor (DOR) ligands in neuropathic pain is not diminished in contrast to other opioid receptor ligands, which lose their effectiveness as analgesics. In this study, we examine whether this effect is related to nerve injury-induced microglial activation. We therefore investigated the influence of minocycline-induced inhibition of microglial activation on the analgesic effects of opioid receptor agonists: morphine, DAMGO, U50,488H, DPDPE, Deltorphin II and SNC80 after chronic constriction injury (CCI) to the sciatic nerve in rats. Pre-emptive and repeated administration of minocycline (30 mg/kg, i.p.) over 7 days significantly reduced allodynia and hyperalgesia as measured on day 7 after CCI. The antiallodynic and antihyperalgesic effects of intrathecally (i.t.) administered morphine (10–20 µg), DAMGO (1–2 µg) and U50,488H (25–50 µg) were significantly potentiated in rats after minocycline, but no such changes were observed after DPDPE (10–20 µg), deltorphin II (1.5–15 µg) and SNC80 (10–20 µg) administration. Additionally, nerve injury-induced down-regulation of all types of opioid receptors in the spinal cord and dorsal root ganglia was not influenced by minocycline, which indicates that the effects of opioid ligands are dependent on other changes, presumably neuroimmune interactions. Our study of rat primary microglial cell culture using qRT-PCR, Western blotting and immunocytochemistry confirmed the presence of mu-opioid receptors (MOR) and kappa-opioid receptors (KOR), further we provide the first evidence for the lack of DOR on microglial cells. In summary, DOR analgesia is different from analgesia induced by MOR and KOR receptors because it does not dependent on injury-induced microglial activation. DOR agonists appear to be the best candidates for new drugs to treat neuropathic pain.     

Opioid receptor selectivity profile change via isosterism for 14-O-substituted naltrexone derivatives

Isosterism is commonly used in drug discovery and development to address stability, selectivity, toxicity, pharmacokinetics, and efficacy issues. A series of 14-O-substituted naltrexone derivatives were identified as potent mu opioid receptor (MOR) antagonists with improved selectivity over the kappa opioid receptor (KOR) and the delta opioid receptor (DOR), compared to naltrexone. Since esters are not metabolically very stable under typical physiological conditions, their corresponding amide analogs were thus synthesized and biologically evaluated. Unlike their isosteres, most of these novel ligands seem to be dually selective for the MOR and the KOR over the DOR. The restricted flexibility of the amide bond linkage might be responsible for their altered selectivity profile. However, the majority of the 14-N-substituted naltrexone derivatives produced marginal or no MOR stimulation in the ³⁵S-GTP[γS] assay, which resembled their ester analogs. The current study thus indicated that the 14-substituted naltrexone isosteres are not bioisosteres since they have distinctive pharmacological profile with the regard to their opioid receptor binding affinity and selectivity.     

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Effect of high dietary copper on weight gain and neuropeptide Y level in the hypothalamus of pigs

An experiment was performed to examine the effect of dietary copper supplementation on weight gain, neuropeptide Y (NPY) concentration and NPY mRNA expression level in the hypothalamus of pigs. Forty-five crossbred pigs were randomly assigned to three groups of 15 pigs, each comprising five replicates of 3 animals. Pigs were allocated to diets that contained 10 mg/kg (as a control), 125 and 250 mg/kg copper as CuSO₄. Live weight gain and feed conversion efficiency was determined at the end of the experiment and five pigs, selected at random from each group, were slaughtered and the hypothalami collected for determination of NPY concentration and NPY mRNA expression level. The results showed that average daily gain (ADG) and average daily feed intake (ADFI) were higher and feed:gain (F:G) ratio was lower in pigs fed the diets with 125 and 250 mg/kg copper (P<0.05), respectively, than in pigs fed a diet with 10 mg/kg copper, but that there was no statistically significant difference in growth performance between animals of the 125 mg/kg and the 250 mg/kg copper groups. Furthermore, pigs fed diets with 125 and 250 mg/kg copper had higher NPY concentrations and NPY mRNA expression levels in their hypothalamus than control animals. The data indicated that 125 and 250 mg/kg copper gave similar responses in terms of weight gain, whilst high dietary copper could enhance NPY concentration and NPY mRNA expression level in the hypothalamus of pigs. High dietary copper appears to increase feed intake and promote weight gain by enhancing NPY concentration and NPY mRNA expression level in the hypothalamus of pigs.     

Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system

Neuropeptide Y (NYP) is a 36 amino acid peptide which shares considerable sequence homology with pancreatic polypeptide and peptide YY. NPY is widely distributed within neurons of the central and peripheral nervous systems, and occurs in mammalian brain in higher concentrations than all other peptides studied to date. Radioimmunoassay studies demonstrated high concentrations of NPY immunoreactivity within many regions of the hypothalamus and within the paraventricular thalamic nucleus, nucleus accumbens, the septum and medial amygdala. These findings correspond with the distribution of NPY containing terminals. Numerous cell bodies containing NPY are located within the cerebral cortex, caudate-putamen, hippocampus, hypothalamus, and nucleus tractus solitarius. Central administration of NPY causes a marked increase in ingestive behaviors, possibly related to the release of NPY from neurons in the arcuate nucleus that innervate the paraventricular nucleus of the hypothalamus. NPY projections from the arcuate nucleus to the medial preoptic area may be related to the central effects of NPY on luteinizing hormone release and sexual behavior. NPY immunoreactive terminals heavily innervated neurons within the amygdala and hypothalamus that are connected to the dorsal vagal complex, suggesting a role of NPY in central autonomic regulation. NPY terminals form a dense plexus around cerebral vessels and are probably responsible for NPY's potent vasoconstrictor effects in the cerebral cortex. Coronary vessels are also innervated heavily by NPY terminals, indicating a role for NPY in the pathogenesis of coronary vasospasm. NPY is present in pheochromocytomas and circulating levels of NPY may prove useful in the diagnosis of pheochromocytoma. Thus, anatomical and physiological studies suggest a varied, but important, function for NPY in mammalian nervous system.     

Differential involvement of the mu and kappa opioid receptors in spatial learning

In order to test the role of mu and kappa opioid receptors (Mu opioid receptor (MOR) and Kappa opioid receptor (KOR)) in hippocampal-dependent spatial learning, we analyzed genetically engineered null mutant mice missing the functional MOR or KOR gene. Compared to wild-type mice, the homozygous MOR null mutants exhibited an impairment in the ultimate level of spatial learning as shown in two distinct tasks, the 8-arm radial-maze and the Morris water-maze. Control behaviors were normal. The learning impairment could be associated with the impairment we found in the maintenance of long-term potentiation in mossy fibers in CA3. In comparison, there was no impairment in spatial learning in our KOR mutants or in mossy fibers (mf) in CA3 region long-term potentiation (LTP). Our work suggests that the MOR may play a positive role in learning and memory by increasing LTP in CA3 neurons.     

用阿片受体放射自显影法研究电针对电惊厥的作用

采用大鼠脑切片的阿片受体结合和放射自显影技术,分析了与电惊厥发作有关的脑内结构及其阿片受体在电针抑制惊厥发作时的变化。图象处理和光密度测定的结果表明,反复电惊厥使尾核、海马、缰核和杏仁核内的阿片受体密度增加。而经电针处理后再给予电惊厥,与电惊厥组相比,海马、缰核和杏仁核内的受体密度降低,在尾核亦显示了受体密度降低的趋势。这一结果为这些结构内的阿片受体参与电惊厥发作和针刺制痫的过程提供了形态学方面的证据。     

Antinociceptive profile of LP1, a non-peptide multitarget opioid ligand

AimsOpioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2 H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects.Main methodsWe explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague–Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug's DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist.Key findingsData obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia.SignificanceLP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain.     

Isobolographic analysis of the opioid-opioid interactions in a tonic and a phasic mouse model of induced nociceptive pain

Background

Opioids have been used for the management of pain and coadministration of two opioids may induce synergism. In a model of tonic pain, the acetic acid writhing test and in a phasic model, the hot plate, the antinociceptive interaction between fentanyl, methadone, morphine, and tramadol was evaluated.

Results

The potency of opioids in the writhing test compared to the hot plate assay was from 2.5 (fentanyl) to 15.5 (morphine) times, respectively. The ED₅₀ was used in a fixed ratio for each of the six pairs of opioid combinations, which, resulted in a synergistic antinociception except for methadone/tramadol and fentanyl/tramadol which were additive, in the hot plate. The opioid antagonists naltrexone, naltrindole and nor-binaltorphimine, suggests that the synergism of morphine combinations are due to the activation of MOR subtypes with partially contribution of DOR and KOR, however fentanyl and methadone combinations are partially due to the activation of MOR and DOR subtypes and KOR lack of participation. The antinociceptive effects of tramadol combinations, are partially due to the activation of MOR, DOR and KOR opioid subtypes.

Conclusion

These results suggets that effectiveness and magnitude of the interactions between opioids are dependent on pain stimulus intensity.     

Expression of delta opioid receptors by splenocytes from SEB-treated mice and effects on phosphorylation of MAP kinase

Delta opioid receptors (DORs) are known to modulate multiple T-cell responses. However, little is known about the expression of these receptors. These studies evaluated the expression of DOR mRNA and protein after a single in vivo exposure to staphylococcal enterotoxin B (SEB). SEB (20 microg, ip) significantly enhanced splenocyte DOR mRNA expression 8 and 24 h after injection. SEB also increased the fractions of the total splenocyte (5 to 20%) and T-cell (8 to 50%) populations expressing DOR protein. In saline-treated animals, DOR relative fluorescence intensity per cell was 11.1 +/- 0.62 units (mean +/- SEM), increasing to 16.1 +/- 1.7 after exposure to SEB. DOR fluorescence intensity significantly increased to 33.5 +/- 2.0 units in a subpopulation of T-cells. Thus, SEB significantly increased DOR expression in vivo, affecting both mRNA and protein levels primarily within the T-cell population. To determine whether T-cell DORs modulate the activity of extracellular-regulated kinases (ERKs), the phosphorylation of ERKs 1 and 2 was studied using splenocytes from SEB-treated mice. At concentrations from 10(-8) to 10(-6) M, [d-Ala(2)-d-Leu(5)]-enkephalin, a selective DOR agonist, significantly inhibited anti-CD3-epsilon-induced phosphorylation of the ERKs. Therefore, the DORs expressed by activated T-cells are capable of attenuating T-cell activation that depends on ERK phosphorylation.