Antibodies raised against the N-terminal sequence of δ opioid receptors blocjed δ-mediated supraspinal antinociception in mice

A polyclonal antiserum directed against the first 16 aminoacids of the N-terminal sequence of the murine δ opioid receptor was raised in rabbits. The intracerebroventricular (i.c.v.) injection to mice of the anti δ receptor IgGs impaired the antinociception produced by DPDPE, [D-Ala²]- Deltorphin II, DADLE and β-endorphin-(1–31) when studied 24 h later in the tail-flick test. Antinociception produced by morphine and DAMGO was fully expressed in mice undergoing this treatment. The selective δ antagonist ICI 174864 (0.8 nmols/mouse, i.c.v.) significantly reduced the antinociceptive activity of opioids to the extent observed after giving the antibodies. ICI 174864 did not decrease further the antinociception that remained after the anti δ receptor serum. The specific binding displayed by 3 nM [³H]-DPDPE was reduced in membranes pre-incubated with the antiserum, whereas no change could be detected for 0.6 nM [³H]-DAMGO labelling μ receptors. This experimental approach revealed the δ component of opioid-evoked supraspinal antinociception in mice.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Atypical responses to morphine in mice: a possible relationship to anorexia nervosa?

According to our previously proposed auto-addiction hypothesis of chronic anorexia nervosa, patients become addicted to an initial period of dieting through endogenous opioid mediated mechanisms. Morphine causes hyperactivity and anorexia in the mouse, symptoms of anorexia nervosa but responses opposite to those of most species including rats and normal human subjects. This suggests that the atypical opioid systems in the mouse may resemble those of the chronic anorexia nervosa patient in contrast to those of most species including the normal human. Characterization of this atypical opioid system may be useful in understanding the pathophysiology of anorexia nervosa.     

A Gi2α antisense oligonucleotide differentiates morphine antinociception,constipation and acute dependence in mice

In the same mice in which the intracerebroventricular (i.c.v.) administration of antisense oligodeoxyribonucleotide (oligo) directed against the G_i2α (but not G_i1α, G_i3α or G_sα) G-protein subunits attenuated i.c.v. morphine-induced antinociception in the tail-flick test, none of the oligos altered naloxone-precipitated jumping (acute dependence). Likewise, none of the oligos significantly altered morphine-induced constipation. Hence, i.c.v. morphine-induced antinociception might be preferentially mediated via transduction pathway(s) different from constipation or acute dependence, offering novel opportunities for drug discovery.     

M₅ muscarinic receptors mediate striatal dopamine activation by ventral tegmental morphine and pedunculopontine stimulation in mice

Opiates, like other addictive drugs, elevate forebrain dopamine levels and are thought to do so mainly by inhibiting GABA neurons near the ventral tegmental area (VTA), in turn leading to a disinhibition of dopamine neurons. However, cholinergic inputs from the laterodorsal (LDT) and pedunculopontine (PPT) tegmental nucleus to the VTA and substantia nigra (SN) importantly contribute, as either LDT or PPT lesions strongly attenuate morphine-induced forebrain dopamine elevations. Pharmacological blockade of muscarinic acetylcholine receptors in the VTA or SN has similar effects. M₅ muscarinic receptors are the only muscarinic receptor subtype associated with VTA and SN dopamine neurons. Here we tested the contribution of M₅ muscarinic receptors to morphine-induced dopamine elevations by measuring nucleus accumbens dopamine efflux in response to intra-VTA morphine infusion using in vivo chronoamperometry. Intra-VTA morphine increased nucleus accumbens dopamine efflux in urethane-anesthetized wildtype mice starting at 10 min after infusion. These increases were absent in M₅ knockout mice and were similarly blocked by pre-treatment with VTA scopolamine in wildtype mice. Furthermore, in wildtype mice electrical stimulation of the PPT evoked an initial, short-lasting increase in striatal dopamine efflux, followed 5 min later by a second prolonged increase in dopamine efflux. In M₅ knockout mice, or following systemic pre-treatment with scopolamine in wildtype mice, the prolonged increase in striatal dopamine efflux was absent. The time course of increased accumbal dopamine efflux in wildtype mice following VTA morphine was consistent with both the prolonged M₅-mediated excitation of striatal dopamine efflux following PPT electrical stimulation and accumbal dopamine efflux following LDT electrical stimulation. Therefore, M₅ receptors appear critical for prolonged PPT excitation of dopamine efflux and for dopamine efflux induced by intra-VTA morphine.     

Molecular mechanisms of allosteric probe dependence in μ opioid receptor

Allostery is one of the most important features of proteins. It greatly contributes to the complexity of life, since it enables possibility of precise tuning of protein function, as well as performing more than one function per protein. Probe dependence is one of the unique features of allostery. It allows a protein to respond differently to the same allosteric modulator when different drugs or transmitters are bound. Unfortunately, allosteric mechanisms are difficult to investigate experimentally. Instead, they can be reproduced artificially in simulations. We simulated in silico a native-like cell membrane fragment with an active-state human μ opioid receptor (MOR) in order to investigate diverse effects of a receptor’s positive allosteric modulator on various agonists. Particular emphasis on native-likeness of the environment was put. We managed to reproduce the experimentally observed effects, which allowed us to take deeper insight into their underlying mechanisms. We found an allosteric pathway in the receptor, leading from the ligand binding site to the intracellular, effector site. We observed that the modulator affected the pathway, inducing different resultant responses for full and partial agonists.     

α-Lipoic acid attenuates x-irradiation-induced oxidative stress in mice

The development of nontoxic but effective radioprotectors is needed because of the increasing risk of human exposure to ionizing radiation. We have reported that α-lipoic acid confers considerable radio-protective effect in mouse tissues when given prior to x-irradiation. In the present study, α-lipoic acid supplementation prior to x-irradiation with 4 and 6 Gy significantly inhibited the radiation-induced decline in total antioxidant capacity (TAC) of plasma. Radiation-induced decline in non-protein sulfhydryl content (NPSH) of different tissues, namely, brain, liver, spleen, kidney, and testis, was also ameliorated significantly at both 4 and 6 Gy doses. Maximal augmentation of radiation-induced protein carbonyl content was observed in spleen followed by brain, kidney, testis, and liver. Maximal protection in terms of carbonyl content was observed in spleen (116%) at 6 Gy dose, and minimal protection was found in liver (22.94%) at 4 Gy dose. Maximal increase in MDA (malondialdehyde) content was observed in brain, followed by testis, spleen, kidney, and liver. Protection by α-lipoic acid pretreatment in terms of MDA content was maximal in brain (51.67%) and minimal in spleen. The findings support the idea that α-lipoic acid is a free-radical scavenger and a potent antioxidant.     

Endogeneous morphine and codeine in mice — effect of muramyl dipeptide

Administration of morphine exerts many effects on the immune system. On the other hand little attention has been paid to the fact, that endogenous morphine and codeine exists in mammals, including man. This raises the question, whether or not endogenous opiate alkaloids play some role in immunoregulation. In addition muramyl-dipeptide (MDP), product of baterial cell wall degradation and a potent immunomdulatory agent exhibits a broad spectrum of effects including effects on CNS functions. The present study investigated whether or not the endogenous levels of morphine and codeine are affected by administration of MDP in mice. Marked variation was found in spleen, brain, small intestine and heart in morphine and codeine concentrations. The intraperitoneal administration of MDP produced a significant increase in tissue morphine levels 30 minutes after injection.     

Etonitazene-induced antinociception in μ1 opioid receptor deficient CXBK mice: Evidence for a role for μ2 receptors in supraspinal antinociception

The prevailing view is that supraspinal μ opioid-mediated antinociception in mice is mediated via the μ₁ subtype. The purpose of the present study was to determine if the highly μ-selective compound etonitazene could produce supraspinal (intracerebroventricular; i.c.v.) antinociception in CXBK mice, which are deficient in brain μ₁, but not μ₂, opioid receptors. CXBK or normal Crl:CD-1 ^®(ICR)BR mice were administered graded doses of etonitazene i.c.v. and 15 min later antinociception was assessed by a standard radiant-heat or 55°C water tail-flick test. Etonitazene produced dose-related antinociception that was blocked by naloxone and by β-FNA (demonstrating a μ opioid mechanism), but not by either ICI-174,864 or naltrindole (demonstrating the lack of involvement of δ opioid receptors). These findings suggest that μ₂ opioid receptors are important contributors to opioid-induced supraspinal antinociception in mice.     

Co-localization of μ and δ opioid receptors on SK-N-SH cells detected by fluorescence microscopy using labeled anti-idiotypic antibodies

Selective fluorescence labeling of opioid receptor subclasses on SK-N-SH cultured cells has been accomplished using labeled polyclonal anti-idiotypic antibodies along with subclass-selective opioid agonists (DPDPE, δ-selective; DAMGO, μ-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of μ- and δ- opioid receptors on SK-N-SH cells has been studied by double labeling fluorescence experiments. In agreement with our own, and other workers', previous observations on NG108-15 cells, a subpopulation of viable cells in asynchronous cultures are labeled. Amon those SK-N-SH cells that are labeled, both subclasses of receptors are seen. On the basis of sequential blocking experiments we interpret our combined results to be consistent with a model where μ- and δ- binding sites reside on different subunits of a multimeric complex.     

Differential response to morphine of the oligomeric state of μ-opioid in the presence of δ-opioid receptors

Prolonged morphine treatment induces extensive desensitization of the μ-opioid receptor (μOR) which is the G-protein-coupled receptor that primarily mediates the cellular response to morphine. To date, the molecular mechanism underlying this process is unknown. Here, we have used live cell fluorescence imaging to investigate whether prolonged morphine treatment affects the physical environment of μOR, or its coupling with G-proteins, in two neuronal cell lines. We find that chronic morphine treatment does not change the amount of enhanced yellow fluorescence protein (eYFP)-tagged μOR on the plasma membrane, and only slightly decreases its association with G-protein subunits. Additionally, morphine treatment does not have a detectable effect on the diffusion coefficient of eYFP-μOR. However, in the presence of another family member, the δ-opioid receptor (δOR), prolonged morphine exposure results in a significant increase in the diffusion rate of μOR. Number and brightness measurements suggest that μOR exists primarily as a dimer that will oligomerize with δOR into tetramers, and morphine promotes the dissociation of these tetramers. To provide a plausible structural context to these data, we used homology modeling techniques to generate putative configurations of μOR-δOR tetramers. Overall, our studies provide a possible rationale for morphine sensitivity.     

Crocetin attenuates inflammation and amyloid-β accumulation in APPsw transgenic mice

Background

Crocetin, an agent derived from saffron, has multiple pharmacological properties, such as neuroprotective, anti-oxidant, and anti-inflammatory actions. These properties might benefit the treatment of Alzheimer’s disease (AD). In the present study, we tested whether crocetin attenuates inflammation and amyloid-β (Aβ) accumulation in APPsw transgenic mice, AD mouse models. Cell viability and the levels of Aβ40 and Aβ42 in HeLa cells stably transfected with Swedish mutant APP751 were evaluated. Mice with Swedish mutant APP751 transgene were used as transgenic mouse models of AD, and were orally administrated with crocetin. Aβ protein and inflammatory cytokines were measured with ELISA. NF-κB and P53 were measured with western blot assay. Learning and memory were analyzed with Morris water maze and novel object recognition tests.

Results

Crocetin significantly reduced Aβ40 and Aβ42 secretion in Hela cells without effecting cell viability. In AD transgenic mice, crocetin significantly reduced the pro-inflammatory cytokines and enhanced anti-inflammatory cytokine in plasma, suppressed NF-κB activation and P53 expression in the hippocampus, decreased Aβ in various brain areas, and improved learning and memory deficits.

Conclusion

Crocetin improves Aβ accumulation-induced learning and memory deficit in AD transgenic mice, probably due to its anti-inflammatory and anti-apoptotic functions.

     

Synthesis and binding affinity of novel mono- and bivalent morphinan ligands for κ, μ, and δ opioid receptors

A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors, and their functional activities were determined at κ and μ receptors in [(35)S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (K(i) values less than 1nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity K(i) values of 0.089nM at the μ receptor and 0.073nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists.     

Are age and sex differences in brain oxytocin receptors related to maternal and infanticidal behavior in naïve mice?

This article is part of a Special Issue “Parental Care”.There is significant variability in the behavioral responses displayed by naïve young and adult mice when first exposed to pups. This variability has been associated with differences in the expression of oxytocin receptors (OXTRs) in the brain in several species. Experiment I investigated the behavioral responses of juvenile, adolescent, and adult CB57BL/6 males and females when first exposed to pups. We found an age increase in maternal females (11% of juveniles, 20% of adolescents, and 50% of young adults), and infanticidal males (0% of juveniles, 30% of adolescents, 44.5% of young adults, and 100% of older adults). Experiment II investigated OXTR density in the brain of juvenile and adult mice. Our results revealed an age decline in the density of OXTR in several brain regions, including the lateral septum, cingulated and posterior paraventricular thalamic nucleus in both males and females. Adult females had higher OXTR density in the ventromedial nucleus/postero-ventral hypothalamus (VMH) and the accessory olfactory bulb (AOB), but lower density in the ventral region of the lateral septum (LSv) than juveniles. Males had lower OXTR density in the anterior olfactory area (AOA) compared to juveniles. No age or sex differences were found in the medial preoptic area, and amygdaloid nuclei, among other brain regions. This study suggests that 1) maturation of parental and infanticidal behavioral responses is not reached until adulthood; 2) the pattern of development of OXTR in the mouse brain is unique, region specific, and differs from that observed in other rodents; 3) either up or down regulation of OXTR in a few brain regions (VMH/AOB/LSv/AOA) might contribute to age or sex differences in parental or infanticidal behavior.     

μ- and δ-opioid receptor antagonists precipitate similar withdrawal phenomena in butorphanol and morphine dependence

The relative involvement of μ- and δ-opioid receptors in the mediation of butorphanol-, as compared to morphine-, dependence was examined with the use of highly selective antagonists at μ- and δ-opioid receptors. Extracellular fluid levels of glutamate (Glu) and aspartate (Asp) were measured within the pontine locus coeruleus following precipitation of withdrawal from dependence on either butorphanol or morphine in conscious Sprague-Dawley rats. Dependence was induced by intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/μl/h), morphine (26 nmol/μl/h) or saline vehicle (1 μl/h) for 3 days by means of an osmotic minipump. Microdialysis probes (2 mm tip) were inserted into the locus coeruleus 24 h before precipitation of withdrawal by i.c.v. injection of either the μ-opioid receptor antagonist,d-Pen-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; 4.8 nmol/5 μl or 48 nmol/5 μl), or the δ-opioid receptor antagonist, naltrindole (17-cyclopropylmethyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2′3′-indolmorphinan hydrochloride; 48 nmol/5 μl or 100 nmol/5 μl). Baseline levels of Glu ranged from 9.59±1.27 to 12.84 ±3.01 μM in the various treatment groups. Level of Asp were similar. Precipitation of withdrawal by CTOP elicited significant increases of Glu and Asp in both morphine- and butorphanol-dependent rats. Maximal increases in Glu of 425% and 258% above baseline levels were elicited in the first 15 min microdialysis sample following i.c.v. injection of CTOP in morphine- and butorphanol-dependent rats, respectively. Behavioral signs of withdrawal were greater in morphine than butorphanol-dependent groups. The i.c.v. treatment with naltrindole elicited increases in Glu and Asp that were similar, although less marked, than those precipitated by CTOP treatment. Administration of naltrindole produced equivalent signs of withdrawal in both morphine- and butorphanol-dependent rats. Withdrawal from dependence on both morphine and butorphanol is characterized by elevations in coerulear levels of excitatory amino acids. Responses elicited following the use of selective μ- and δ-opioid receptor antagonists to precipitate withdrawal suggest that the role played by these receptors in mediation of the signs and symptoms of withdrawal do not differ greatly between butorphanol- and morphine-dependent rats.     

Ei24-deficiency attenuates protein kinase Cα signaling and skin carcinogenesis in mice

Etoposide-induced gene 24 (Ei24) is a p53 target gene that inhibits growth, induces apoptosis and autophagy, as well as suppresses breast cancer. To evaluate the role of Ei24 in in vivo tumorigenesis, we generated an Ei24-deficient mouse model. Here, we report that, although Ei24 homozygous knockout mice are embryonic lethal, Ei24 heterozygous null mice are attenuated to DMBA/TPA-induced carcinogenesis with regard to the number and size of tumors but not the incidence. Ei24 contains a functional consensus motif, named as an R motif that is highly analogous to amino acids 105-110 of RINCK1, an E3 ligase for protein kinase C (PKC) proteins. We found that Ei24 stabilizes PKCαvia RINCK degradation and competition with RINCK for binding with the C1a domain of PKCα. We also found that Ei24 contributes to PKCα-mediated transactivation of EGFR by promoting PKCα membrane localization and interaction with EGFR. Finally, using Oncomine database we show that Ei24 and EGFR are upregulated in some subsets of human HNSCC. These results suggest that Ei24 is a regulator of the RINCK1-PKCα-EGFR signaling pathway in the development of skin-cancer.