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Sample records for acting mu-opioid receptor

  1. Naloxegol: First oral peripherally acting mu opioid receptor antagonists for opioid-induced constipation

    PubMed Central

    Anantharamu, Tejus; Sharma, Sushil; Gupta, Ajay Kumar; Dahiya, Navdeep; Singh Brashier, Dick B.; Sharma, Ashok Kumar

    2015-01-01

    Opioid-induced constipation (OIC) is one of the most troublesome and the most common effects of opioid use leading to deterioration in quality of life of the patients and also has potentially deleterious repercussions on adherence and compliance to opioid therapy. With the current guidelines advocating liberal use of opioids by physicians even for non-cancer chronic pain, the situation is further complicated as these individuals are not undergoing palliative care and hence there cannot be any justification to subject these patients to the severe constipation brought on by opioid therapy which is no less debilitating than the chronic pain. The aim in these patients is to prevent the opioid-induced constipation but at the same time allow the analgesic activity of opioids. Many drugs have been used with limited success but the most specific among them were the peripherally acting mu opioid receptor antagonists (PAMORA). Methylnaltrexone and alvimopan were the early drugs in this group but were not approved for oral use in OIC. However naloxegol, the latest PAMORA has been very recently approved as the first oral drug for OIC. This article gives an overview of OIC, its current management and more specifically the development and approval of naloxegol, including pharmacokinetics, details of various clinical trials, adverse effects and its current status for the management of OIC. PMID:26312011

  2. Mu opioid receptor binding sites in human brain

    SciTech Connect

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand (/sup 3/H)DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of (/sup 3/H)DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas.

  3. Mu Opioid Receptor Actions in the Lateral Habenula

    PubMed Central

    Margolis, Elyssa B.; Fields, Howard L.

    2016-01-01

    Increased activity of lateral habenula (LHb) neurons is correlated with aversive states including pain, opioid abstinence, rodent models of depression, and failure to receive a predicted reward. Agonists at the mu opioid receptor (MOR) are among the most powerful rewarding and pain relieving drugs. Injection of the MOR agonist morphine directly into the habenula produces analgesia, raising the possibility that MOR acts locally within the LHb. Consequently, we examined the synaptic actions of MOR agonists in the LHb using whole cell patch clamp recording. We found that the MOR selective agonist DAMGO inhibits a subset of LHb neurons both directly and by inhibiting glutamate release onto these cells. Paradoxically, DAMGO also presynaptically inhibited GABA release onto most LHb neurons. The behavioral effect of MOR activation will thus depend upon both the level of intrinsic neuronal activity in the LHb and the balance of activity in glutamate and GABA inputs to different LHb neuronal populations. PMID:27427945

  4. Mu Opioid Receptor Actions in the Lateral Habenula.

    PubMed

    Margolis, Elyssa B; Fields, Howard L

    2016-01-01

    Increased activity of lateral habenula (LHb) neurons is correlated with aversive states including pain, opioid abstinence, rodent models of depression, and failure to receive a predicted reward. Agonists at the mu opioid receptor (MOR) are among the most powerful rewarding and pain relieving drugs. Injection of the MOR agonist morphine directly into the habenula produces analgesia, raising the possibility that MOR acts locally within the LHb. Consequently, we examined the synaptic actions of MOR agonists in the LHb using whole cell patch clamp recording. We found that the MOR selective agonist DAMGO inhibits a subset of LHb neurons both directly and by inhibiting glutamate release onto these cells. Paradoxically, DAMGO also presynaptically inhibited GABA release onto most LHb neurons. The behavioral effect of MOR activation will thus depend upon both the level of intrinsic neuronal activity in the LHb and the balance of activity in glutamate and GABA inputs to different LHb neuronal populations. PMID:27427945

  5. Alcohol addiction and the mu-opioid receptor.

    PubMed

    Berrettini, Wade

    2016-02-01

    Alcohol addiction is one of the most common and devastating diseases in the world. Given the tremendous heterogeneity of alcohol addicted individuals, it is unlikely that one medication will help nearly all patients. Thus, there is a clear need to develop predictors of response to existing medications. Naltrexone is a mu-opioid receptor antagonist which has been approved in the United States for treatment of alcohol addiction since 1994. It has limited efficacy, in part due to noncompliance, but many patients do not respond despite high levels of compliance. There are reports that a mis-sense single nucleotide polymorphism (rs179919 or A118G) in the mu-opioid receptor gene predicts a favorable response to naltrexone if an individual carries a 'G' allele. This chapter will review the evidence for this hypothesis. The data are promising that the 'G' allele predisposes to a beneficial naltrexone response among alcohol addicted persons, but additional research is needed to prove this hypothesis in prospective clinical trials. PMID:26226591

  6. Mu opioid receptor polymorphism, early social adversity, and social traits.

    PubMed

    Carver, Charles S; Johnson, Sheri L; Kim, Youngmee

    2016-10-01

    A polymorphism in the mu opioid receptor gene OPRM1 (rs1799971) has been investigated for its role in sensitivity to social contexts. Evidence suggests that the G allele of this polymorphism is associated with higher levels of sensitivity. This study tested for main effects of the polymorphism and its interaction with a self-report measure of childhood adversity as an index of negative environment. Outcomes were several personality measures relevant to social connection. Significant interactions were obtained, such that the negative impact of childhood adversity on personality was greater among G carriers than among A homozygotes on measures of agreeableness, interdependence, anger proneness, hostility, authentic pride, life engagement, and an index of (mostly negative) feelings coloring one's world view. Findings support the role of OPRM1 in sensitivity to negative environments. Limitations are noted, including the lack of a measure of advantageous social environment to assess sensitivity to positive social contexts. PMID:26527429

  7. Mu opioid receptors in developing human spinal cord

    PubMed Central

    RAY, SUBRATA BASU; WADHWA, SHASHI

    1999-01-01

    The distribution of mu opioid receptors was studied in human fetal spinal cords between 12–13 and 24–25 wk gestational ages. Autoradiographic localisation using [3H] DAMGO revealed the presence of mu receptors in the dorsal horn at all age groups with a higher density in the superficial laminae (I–II). A biphasic expression was noted. Receptor density increased in the dorsal horn, including the superficial laminae, between 12–13 and 16–17 wk. This could be associated with a spurt in neurogenesis. The density increased again at 24–25 wk in laminae I–II which resembled the adult pattern of distribution. A dramatic proliferation of cells was noted from the region of the ventricular zone between 16–17 and 24–25 wk. These were considered to be glial cells from their histological features. Mu receptor expression was noted over a large area of the spinal cord including the lateral funiculus at 24–25 wk. This may be due to receptor expression by glial cells. The study presents evidence of mu receptor expression by both neurons and glia during early development of human spinal cord. PMID:10473288

  8. Mu-opioid receptors modulate the stability of dendritic spines.

    PubMed

    Liao, Dezhi; Lin, Hang; Law, Ping Yee; Loh, Horace H

    2005-02-01

    Opioids classically regulate the excitability of neurons by suppressing synaptic GABA release from inhibitory neurons. Here, we report a role for opioids in modulating excitatory synaptic transmission. By activating ubiquitously clustered mu-opioid receptor (MOR) in excitatory synapses, morphine caused collapse of preexisting dendritic spines and decreased synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Meanwhile, the opioid antagonist naloxone increased the density of spines. Chronic treatment with morphine decreased the density of dendritic spines even in the presence of Tetrodotoxin, a sodium channel blocker, indicating that the morphine's effect was not caused by altered activity in neural network through suppression of GABA release. The effect of morphine on dendritic spines was absent in transgenic mice lacking MORs and was blocked by CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2), a mu-receptor antagonist. These data together with others suggest that endogenous opioids and/or constitutive activity of MORs participate in maintaining normal morphology and function of spines, challenging the classical model of opioids. Abnormal alteration of spines may occur in drug addiction when opioid receptors are overactivated by exogenous opiates. PMID:15659552

  9. The rewarding properties of MDMA are preserved in mice lacking mu-opioid receptors.

    PubMed

    Robledo, Patricia; Mendizabal, Victoria; Ortuño, Jordi; de la Torre, Rafael; Kieffer, Brigitte L; Maldonado, Rafael

    2004-08-01

    The involvement of mu-opioid receptors in the rewarding properties of MDMA was explored in mu-opioid receptor knockout mice using the conditioning place preference paradigm. The associated release of dopamine in the nucleus accumbens was investigated by in vivo microdialysis. A significant rewarding effect of MDMA (10 mg/kg, i.p.) was observed in both wild-type and mu-opioid receptor knockout mice. MDMA (10 mg/kg, i.p.) also induced similar increases in dopamine and decreases in 3,4-dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens dialysates of both wild-type and mu-opioid receptor knockout mice. No significant differences in basal levels of dopamine, 3,4-dihydroxyphenylacetic or homovanillic acids between wild-type and mu-opioid receptor knockout mice were observed. In summary, the present results suggest that, in contrast to what has been reported for other drugs of abuse such as opioids, ethanol, nicotine and Delta(9)-tetrahydrocannabinol, mu-opioid receptors do not play a major role in the rewarding properties of MDMA. These differences could be due to distinct mechanisms controlling dopamine release in the nucleus accumbens and suggest that the effects of MDMA on dopaminergic neurons are independent of micro -opioid receptors. PMID:15255997

  10. Prolonged central mu-opioid receptor occupancy after single and repeated nalmefene dosing.

    PubMed

    Ingman, Kimmo; Hagelberg, Nora; Aalto, Sargo; Någren, Kjell; Juhakoski, Auni; Karhuvaara, Sakari; Kallio, Antero; Oikonen, Vesa; Hietala, Jarmo; Scheinin, Harry

    2005-12-01

    The opioid antagonist nalmefene offers an alternative to traditional pharmacological treatments for alcoholism. The present study was designed to investigate the relationship between nalmefene plasma concentration and central mu-opioid receptor occupancy after a clinically effective dose (20 mg, orally). Pharmacokinetics and mu-opioid receptor occupancy of nalmefene after single and repeated dosing over 7 days was studied in 12 healthy subjects. Serial blood samples were obtained after both dosings, and pharmacokinetic parameters for nalmefene and main metabolites were determined. Central mu-opioid receptor occupancy of nalmefene was measured with positron emission tomography (PET) and [(11)C]carfentanil at four time points (3, 26, 50, 74 h) after both dosings. Nalmefene was rapidly absorbed in all subjects. The mean t(1/2) of nalmefene was 13.4 h after single and repeated dosing. The accumulation of nalmefene and its main metabolites in plasma during the repeated dosing period was as expected for a drug with linear pharmacokinetics, and steady-state was reached for all analytes. Both nalmefene dosings resulted in a very high occupancy at mu-opioid receptors (87-100%), and the decline in the occupancy was similar after both dosings but clearly slower than the decline in the plasma concentration of nalmefene or metabolites. High nalmefene occupancy (83-100%) persisted at 26 h after the dosings. The prolonged mu-opioid receptor occupancy by nalmefene indicates slow dissociation of the drug from mu-opioid receptors. These results support the rational of administering nalmefene when needed before alcohol drinking, and they additionally suggest that a high mu-opioid receptor occupancy can be maintained when nalmefene is taken once daily. PMID:15956985

  11. The presence of the mu-opioid receptor in the isthmus of mare oviduct.

    PubMed

    Desantis, S; Albrizio, M; Ventriglia, G; Deflorio, M; Guaricci, A C; Minoia, R; De Metrio, G

    2008-05-01

    The presence of the mu-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (N-glycosidase F and O-glycosidase) and chemical (beta-elimination) treatments. Immunoblotting analysis showed that the mu-opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by O-glycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the mu-opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galbeta(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the mu-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the mu-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after N-glycosidase F treatment, whereas it was preserved in the apical region after beta-elimination. Most non-ciliated cells expressed the mu-opioid receptor with both N- and O-linked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and beta-elimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the mu-opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the mu-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the mu-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective

  12. Shadows across mu-Star? Constitutively active mu-opioid receptors revisited.

    PubMed

    Connor, Mark

    2009-04-01

    Constitutively active mu-opioid receptors (mu* receptors) are reported to be formed following prolonged agonist treatment of cells or whole animals. mu* receptors signal in the absence of activating ligand and a blockade of mu* activation of G-proteins by naloxone and naltrexone has been suggested to underlie the profound withdrawal syndrome precipitated by these antagonists in vivo. In this issue of the Journal, Divin et al. examined whether treatment of C6 glioma cells with mu-opioid receptor agonists produced constitutively active mu-opioid receptors or other commonly reported adaptations to prolonged agonist treatment. Adenylyl cyclase superactivation was readily apparent following agonist treatment but there was no evidence of the formation of constitutively active mu-opioid receptors. This result challenges the notion that prolonged agonist exposure inevitably produces mu* receptors, and is consistent with many studies of adaptations in neurons produced by chronic agonist treatment. The investigators provide no explanation of their failure to see mu* receptors in C6 cells, but this is perhaps understandable because the molecular nature of mu* receptors remains elusive, and the precise mechanisms that lead to their formation are unknown. Without knowing exactly what mu* receptors are, how they are formed and how they signal, understanding their role in cellular adaptations to prolonged opioid treatment will remain impossible. Studies such as this should refocus attention on establishing the molecular mechanisms that underlie that phenomenon of mu* receptors. PMID:19368530

  13. Renal mu opioid receptor mechanisms in regulation of renal function in rats.

    PubMed

    Kapusta, D R; Jones, S Y; DiBona, G F

    1991-07-01

    Studies were performed in pentobarbital anesthetized Sprague-Dawley rats to determine whether mu opioid receptor agonists produce changes in renal function via intrarenal mechanisms. Left renal artery infusion of isotonic saline vehicle or the selective mu opioid receptor agonist, dermorphin (0.5 nmol/kg/min), did not alter mean arterial pressure or heart rate. In contrast, left renal artery dermorphin administration produced a significant decrease in left kidney urinary flow rate and sodium excretion without altering glomerular filtration rate or effective renal plasma flow; function of the right kidney was unaffected. Pretreatment of the left kidney with the opioid receptor antagonist naloxone, 50 micrograms/kg into left renal artery, prevented changes in urinary flow rate and sodium excretion induced by subsequent left renal artery dermorphin administration. Prior bilateral renal denervation abolished the antidiuretic and antinatriuretic responses to left renal artery dermorphin administration. These results suggest that mu opioid receptor agonists participate in the process of renal tubular sodium and water reabsorption via an intrarenal action that is dependent on an interaction with renal sympathetic nerves. This may occur via an action of mu opioid receptor agonists to facilitate the nerve terminal release and/or the direct tubular action of norepinephrine to affect renal tubular sodium and water reabsorption. PMID:1677034

  14. Redoubling the ring size of an endomorphin-2 analog transforms a centrally acting mu-opioid receptor agonist into a pure peripheral analgesic.

    PubMed

    Piekielna, Justyna; De Marco, Rossella; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tömböly, Csaba; Artali, Roberto; Janecka, Anna

    2016-05-01

    The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2 . The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 )2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016. PMID:27038094

  15. Interactions between cannabinoid receptor agonists and mu opioid receptor agonists in rhesus monkeys discriminating fentanyl.

    PubMed

    Maguire, David R; France, Charles P

    2016-08-01

    Cannabinoid receptor agonists such as delta-9-tetrahydrocannabinol (Δ(9)-THC) enhance some (antinociceptive) but not other (positive reinforcing) effects of mu opioid receptor agonists, suggesting that cannabinoids might be combined with opioids to treat pain without increasing, and possibly decreasing, abuse. The degree to which cannabinoids enhance antinociceptive effects of opioids varies across drugs insofar as Δ(9)-THC and the synthetic cannabinoid receptor agonist CP55940 increase the potency of some mu opioid receptor agonists (e.g., fentanyl) more than others (e.g., nalbuphine). It is not known whether interactions between cannabinoids and opioids vary similarly for other (abuse-related) effects. This study examined whether Δ(9)-THC and CP55940 differentially impact the discriminative stimulus effects of fentanyl and nalbuphine in monkeys (n=4) discriminating 0.01mg/kg of fentanyl (s.c.) from saline. Fentanyl (0.00178-0.0178mg/kg) and nalbuphine (0.01-0.32mg/kg) dose-dependently increased drug-lever responding. Neither Δ(9)-THC (0.032-1.0mg/kg) nor CP55940 (0.0032-0.032mg/kg) enhanced the discriminative stimulus effects of fentanyl or nalbuphine; however, doses of Δ(9)-THC and CP55940 that shifted the nalbuphine dose-effect curve markedly to the right and/or down were less effective or ineffective in shifting the fentanyl dose-effect curve. The mu opioid receptor antagonist naltrexone (0.032mg/kg) attenuated the discriminative stimulus effects of fentanyl and nalbuphine similarly. These data indicate that the discriminative stimulus effects of nalbuphine are more sensitive to attenuation by cannabinoids than those of fentanyl. That the discriminative stimulus effects of some opioids are more susceptible to modification by drugs from other classes has implications for developing maximally effective therapeutic drug mixtures with reduced abuse liability. PMID:27184925

  16. Involvement of peripheral mu opioid receptors in scratching behavior in mice.

    PubMed

    Yamamoto, Atsuki; Sugimoto, Yukio

    2010-12-15

    Pruritus is a common adverse effect of opioid treatment. However, the mechanism by which pruritus is induced by opioid administration is unclear. In this study, we examined the effects of the intradermal injection of loperamide, a peripherally restricted opioid receptor agonist, on the itch sensation. When injected intradermally into the rostral part of the back in mice, loperamide elicited scratching behavior. We also examined the effects of the selective mu opioid receptor agonist [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate (DAMGO), the selective delta opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE), and the selective kappa opioid receptor agonist U-50488H on scratching behavior in mice in order to determine which subtype is involved in opioid-induced pruritus. Following intradermal injection into the rostral part of the back in mice, DAMGO elicited scratching behavior, while DPDPE and U-50488H did not. This suggests that peripheral mu opioid activation elicits the itch sensation. Next, we focused on the treatment of opioid-induced itch sensation without central adverse effects. Naloxone methiodide is a peripherally restricted opioid receptor antagonist. In the present study, naloxone methiodide significantly suppressed scratching behavior induced by loperamide and DAMGO. These findings suggest that mu opioid receptors play a primary role in peripheral pruritus and that naloxone methiodide may represent a possible remedy for opioid-induced itching. PMID:20863827

  17. Genomic structure analysis of promoter sequence of a mouse mu opioid receptor gene.

    PubMed Central

    Min, B H; Augustin, L B; Felsheim, R F; Fuchs, J A; Loh, H H

    1994-01-01

    We have isolated mouse mu opioid receptor genomic clones (termed MOR) containing the entire amino acid coding sequence corresponding to rat MOR-1 cDNA, including additional 5' flanking sequence. The mouse MOR gene is > 53 kb long, and the coding sequence is divided by three introns, with exon junctions in codons 95 and 213 and between codons 386 and 387. The first intron is > 26 kb, the second is 0.8 kb, and the third is > 12 kb. Multiple transcription initiation sites were observed, with four major sites confirmed by 5' rapid amplification of cDNA ends and RNase protection located between 291 and 268 bp upstream of the translation start codon. Comparison of the 5' flanking sequence with a transcription factor database revealed putative cis-acting regulatory elements for transcription factors affected by cAMP, as well as those involved in the action of gluco- and mineralocorticoids, cytokines, and immune-cell-specific factors. Images PMID:8090773

  18. Mu Opioids and Their Receptors: Evolution of a Concept

    PubMed Central

    Pan, Ying-Xian

    2013-01-01

    Opiates are among the oldest medications available to manage a number of medical problems. Although pain is the current focus, early use initially focused upon the treatment of dysentery. Opium contains high concentrations of both morphine and codeine, along with thebaine, which is used in the synthesis of a number of semisynthetic opioid analgesics. Thus, it is not surprising that new agents were initially based upon the morphine scaffold. The concept of multiple opioid receptors was first suggested almost 50 years ago (Martin, 1967), opening the possibility of new classes of drugs, but the morphine-like agents have remained the mainstay in the medical management of pain. Termed mu, our understanding of these morphine-like agents and their receptors has undergone an evolution in thinking over the past 35 years. Early pharmacological studies identified three major classes of receptors, helped by the discovery of endogenous opioid peptides and receptor subtypes—primarily through the synthesis of novel agents. These chemical biologic approaches were then eclipsed by the molecular biology revolution, which now reveals a complexity of the morphine-like agents and their receptors that had not been previously appreciated. PMID:24076545

  19. Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

    PubMed Central

    Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

    2013-01-01

    Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

  20. Powerful inhibitory action of mu opioid receptors (MOR) on cholinergic interneuron excitability in the dorsal striatum.

    PubMed

    Ponterio, G; Tassone, A; Sciamanna, G; Riahi, E; Vanni, V; Bonsi, P; Pisani, A

    2013-12-01

    Cholinergic interneurons (ChIs) of dorsal striatum play a key role in motor control and in behavioural learning. Neuropeptides regulate cholinergic transmission and mu opioid receptor (MOR) activation modulates striatal acetylcholine release. However, the mechanisms underlying this effect are yet uncharacterized. Here, we examined the electrophysiological responses of ChIs to the selective MOR agonist, DAMGO {[D-Ala2-MePhe4-Gly(ol)5] enkephalin}. We observed a robust, dose-dependent inhibition of spontaneous firing activity (0.06-3 μM) which was reversible upon drug washout and blocked by the selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) (1 μM). Voltage-clamp analysis of the reversal potential of the DAMGO effect did not provide univocal results, indicating the involvement of multiple membrane conductances. The MOR-dependent effect persisted in the presence of GABAA and ionotropic glutamate receptor antagonists, ruling out an indirect effect. Additionally, it depended upon G-protein activation, as it was prevented by intrapipette GDP-β-S. Because D2 dopamine receptors (D2R) and MOR share a common post-receptor signalling pathway, occlusion experiments were performed with maximal doses of both D2R and MOR agonists. The D2R agonist quinpirole decreased spike discharge, which was further reduced by adding DAMGO. Then, D2R or MOR antagonists were used to challenge the response to the respective agonists, DAMGO or quinpirole. No cross-effect was observed, suggesting that the two receptors act independently. Our findings demonstrate a postsynaptic inhibitory modulation by MOR on ChIs excitability. Such opioidergic regulation of cholinergic transmission might contribute to shape information processing in basal ganglia circuits, and represent a potential target for pharmacological intervention. PMID:23891638

  1. Real-time imaging of Mu opioid receptors by total internal reflection fluorescence microscopy

    PubMed Central

    Roman-Vendrell, Cristina; Yudowski, Guillermo Ariel

    2016-01-01

    Receptor trafficking and signaling are intimately linked, especially in the Mu opioid receptor (MOR) where ligand dependent endocytosis and recycling have been associated to opioid tolerance and dependence. Ligands of the Mu opioid receptor (MOR) can induce receptor endocytosis and recycling within minutes of exposure in heterologous systems and cultured neurons. Endocytosis removes desensitized receptors after their activation from the plasma membrane, while recycling promotes resensitization by delivering functional receptors to the cell surface. These rapid mechanisms can escape traditional analytical methods where only snapshots are obtained from highly dynamic events. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool that can be used to investigate, in real-time, surface trafficking events at the single molecule level. The restricted excitation of fluorophores located at or near the plasma membrane in combination with high sensitivity quantitative cameras, makes it possible to record and analyze individual endocytic and recycling event in real time. In this chapter, we describe a TIRF microscopy protocol to investigate in real time, the ligand dependent MOR trafficking in Human Embryonic Kidney 293 cells and dissociated striatal neuronal cultures. This approach can provide unique spatio-temporal resolution to understand the fundamental events controlling MOR trafficking at the plasma membrane. PMID:25293317

  2. Human Mu Opioid Receptor (OPRM1A118G) polymorphism is associated with brain mu- opioid receptor binding potential in smokers

    SciTech Connect

    Ray, R.; Logan, J.; Ray, R.; Ruparel, K.; Newberg, A.; Wileyto, E.P.; Loughead, J.W.; Divgi, C.; Blendy, J.A.; Logan, J.; Zubieta, J.-K.; Lerman, C.

    2011-04-15

    Evidence points to the endogenous opioid system, and the mu-opioid receptor (MOR) in particular, in mediating the rewarding effects of drugs of abuse, including nicotine. A single nucleotide polymorphism (SNP) in the human MOR gene (OPRM1 A118G) has been shown to alter receptor protein level in preclinical models and smoking behavior in humans. To clarify the underlying mechanisms for these associations, we conducted an in vivo investigation of the effects of OPRM1 A118G genotype on MOR binding potential (BP{sub ND} or receptor availability). Twenty-two smokers prescreened for genotype (12 A/A, 10 */G) completed two [{sup 11}C] carfentanil positron emission tomography (PET) imaging sessions following overnight abstinence and exposure to a nicotine-containing cigarette and a denicotinized cigarette. Independent of session, smokers homozygous for the wild-type OPRM1 A allele exhibited significantly higher levels of MOR BP{sub ND} than smokers carrying the G allele in bilateral amygdala, left thalamus, and left anterior cingulate cortex. Among G allele carriers, the extent of subjective reward difference (denicotinized versus nicotine cigarette) was associated significantly with MOR BP{sub ND} difference in right amygdala, caudate, anterior cingulate cortex, and thalamus. Future translational investigations can elucidate the role of MORs in nicotine addiction, which may lead to development of novel therapeutics.

  3. Partial purification of the mu opioid receptor irreversibly labeled with (/sup 3/H)b-funaltrexamine

    SciTech Connect

    Liu-Chen, L.Y.; Phillips, C.A.; Tam, S.W.

    1986-03-01

    The mu opioid receptor in bovine striatal membranes was specifically and irreversibly labeled by incubation with 5 nM (/sup 3/H)..beta..-funaltrexamine (approx.-FNA) at 37/sup 0/C for 90 min in the presence of 100 mM NaCl. The specific and irreversible binding of (/sup 3/H)..beta..-FNA as defined by that blocked by 1 /sup +/M naloxone was about 60% of total irreversible binding. The specific irreversible binding was saturable, stereospecific, time-, temperature, and tissue-dependent. Mu opioid ligands were much more potent than delta or kappa ligands in inhibiting the specific irreversible labeling. SDS polyacrylamide gel electrophoresis of solubilized membranes in the presence of 2-mercaptoethanol yielded a major radiolabeled broad band of MW 68-97K daltons, characteristic of a glycoprotein band. This band was not observed in membranes labeled in the presence of excess unlabeled naloxone. The glycoprotein nature of the (/sup 3/H)..beta..-FNA-labeled opioid receptor was confirmed by its binding to a wheat germ agglutinin-Sepharose column and its elution with N-acetylglucosamine.

  4. Quantitative autoradiography of (/sup 3/H)CTOP binding to mu opioid receptors in rat brain

    SciTech Connect

    Hawkins, K.N.; Knapp, R.J.; Gehlert, D.R.; Lui, G.K.; Yamamura, M.S.; Roeske, L.C.; Hruby, V.J.; Yamamura, H.I.

    1988-01-01

    (/sup 3/H)H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ((/sup 3/H)CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with (/sup 3/H)CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited (/sup 3/H)CTOP binding with high affinity (IC50 values of 0.2-2.4nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69,593 were very weak inhibitors of (/sup 3/H)CTOP binding. Light microscopic autoradiography of (/sup 3/H)CTOP binding sites revealed regions of high density and regions of moderate labeling. The cerebral cortex showed a low density of (/sup 3/H)CTOP binding.

  5. Detecting the mu opioid receptor in brain following SDS-PAGE with multiple approaches

    PubMed Central

    Huang, Peng; Liu-Chen, Lee-Yuan

    2013-01-01

    In general, it has been difficult to obtain antibodies which are useful for immunoblotting of endogenous seven-transmembrane receptors (7TMRs) despite the claims made by many companies on commercially available antibodies. In this review, we will use the mu opioid receptor (MOPR) in brain as an example to underscore the importance of using knock-out (K/O) mice and multiple independent approaches (ligand affinity-labeling, receptor phosphorylation and immunoblotting) in identifying 7TMRs following sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). The rigor and convergence of pharmacological and biochemical data provide confidence on the unequivocal identification of MOPR. The distinct relative molecular masses (Mr’s) and band patterns are largely due to variations in the extent of N-glycosylation in different cell lines, brain regions and species. PMID:19482639

  6. Binding characteristics of [3H]14-methoxymetopon, a high affinity mu-opioid receptor agonist.

    PubMed

    Spetea, Mariana; Tóth, Fanni; Schütz, Johannes; Otvös, Ferenc; Tóth, Géza; Benyhe, Sandor; Borsodi, Anna; Schmidhammer, Helmut

    2003-07-01

    The highly potent micro -opioid receptor agonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the micro -opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPgammaS binding was inhibited by naloxone and selective micro -opioid antagonists, indicating a micro -opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level. PMID:12887410

  7. Fourteen. beta. -(bromoacetamido)morphine irreversibly labels. mu. opioid receptors in rat brain membranes

    SciTech Connect

    Bidlack, J.M.; Frey, D.K.; Seyed-Mozaffari, A.; Archer, S. )

    1989-05-16

    The binding properties of 14{beta}-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM ({sup 3}H)(D-Ala{sup 2},(Me)Phe{sup 4},Gly(ol){sup 5})enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the {mu} binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The {mu} receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the {delta}-selective peptide ({sup 3}H)(D-penicillamine{sup 2},D-penicillamine{sup 5})enkephalin (DPDPE) and (-)-({sup 3}H)bremazocine in the presence of {mu} and {delta} blockers, selective for {kappa} binding sites. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the {mu}s site did not afford protection. These studies have demonstrated that when a disulfide bond at or near {mu} opioid binding sites was reduced, BAM could then alkylate this site, resulting in the specific irreversible labeling of {mu} opioid receptors.

  8. Human population genetic structure detected by pain-related mu opioid receptor gene polymorphisms

    PubMed Central

    López Soto, Eduardo Javier; Catanesi, Cecilia Inés

    2015-01-01

    Several single nucleotide polymorphisms (SNPs) in the Mu Opioid Receptor gene (OPRM1) have been identified and associated with a wide variety of clinical phenotypes related both to pain sensitivity and analgesic requirements. The A118G and other potentially functional OPRM1 SNPs show significant differences in their allele distributions among populations. However, they have not been properly addressed in a population genetic analysis. Population stratification could lead to erroneous conclusions when they are not taken into account in association studies. The aim of our study was to analyze OPRM1 SNP variability by comparing population samples of the International Hap Map database and to analyze a new population sample from the city of Corrientes, Argentina. The results confirm that OPRM1 SNP variability differs among human populations and displays a clear ancestry genetic structure, with three population clusters: Africa, Asia, and Europe-America. PMID:26273217

  9. Analgesic effect of interferon-alpha via mu opioid receptor in the rat.

    PubMed

    Jiang, C L; Son, L X; Lu, C L; You, Z D; Wang, Y X; Sun, L Y; Cui, R Y; Liu, X Y

    2000-03-01

    Using the tail-flick induced by electro-stimulation as a pain marker, it was found that pain threshold (PT) was significantly increased after injecting interferon-alpha (IFN alpha) into the lateral ventricle of rats. This effect was dosage-dependent and abolished by monoclonal antibody (McAb) to IFN alpha. Naloxone could inhibit the analgesic effect of IFN alpha, suggesting that the analgesic effect of IFN alpha be related to the opioid receptors. Beta-funaltrexamine (beta-FNA), the mu specific receptor antagonist could completely block the analgesic effect of IFN alpha. The selective delta-opioid receptor antagonist, ICI174,864 and the kappa-opioid receptor antagonist, nor-BNI both failed to prevent the analgesic effect of IFN alpha. IFN alpha could significantly inhibit the production of the cAMP stimulated by forskolin in SK-N-SH cells expressing the mu-opioid receptor, not in NG108-15 cells expressing the delta-opioid receptor uniformly. The results obtained provide further evidence for opioid activity of IFN alpha and suggest that this effect is mediated by central opioid receptors of the mu subtype. The evidence is consistent with the hypothesis that multiple actions of cytokines, such as immunoregulatory and neuroregulatory effects, might be mediated by distinct domains of cytokines interacting with different receptors. PMID:10676852

  10. Muscarinic acetylcholine receptor modulation of mu (mu) opioid receptors in adult rat sphenopalatine ganglion neurons.

    PubMed

    Margas, Wojciech; Mahmoud, Saifeldin; Ruiz-Velasco, Victor

    2010-01-01

    The sphenopalatine ganglion (SPG) neurons represent the parasympathetic branch of the autonomic nervous system involved in controlling cerebral blood flow. In the present study, we examined the coupling mechanism between mu (mu) opioid receptors (MOR) and muscarinic acetylcholine receptors (mAChR) with Ca(2+) channels in acutely dissociated adult rat SPG neurons. Successful MOR activation was recorded in approximately 40-45% of SPG neurons employing the whole cell variant of the patch-clamp technique. In addition, immunofluorescence assays indicated that MOR are not expressed in all SPG neurons while M(2) mAChR staining was evident in all neurons. The concentration-response relationships generated with morphine and [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) showed IC(50) values of 15.2 and 56.1 nM and maximal Ca(2+) current inhibition of 26.0 and 38.7%, respectively. Activation of MOR or M(2) mAChR with morphine or oxotremorine-methiodide (Oxo-M), respectively, resulted in voltage-dependent inhibition of Ca(2+) currents via coupling with Galpha(i/o) protein subunits. The acute prolonged exposure (10 min) of neurons to morphine or Oxo-M led to the homologous desensitization of MOR and M(2) mAChR, respectively. The prolonged stimulation of M(2) mAChR with Oxo-M resulted in heterologous desensitization of morphine-mediated Ca(2+) current inhibition, and was sensitive to the M(2) mAChR blocker methoctramine. On the other hand, when the neurons were exposed to morphine or DAMGO for 10 min, heterologous desensitization of M(2) mAChR was not observed. These results suggest that in rat SPG neurons activation of M(2) mAChR likely modulates opioid transmission in the brain vasculature to adequately maintain cerebral blood flow. PMID:19889856

  11. Systematic analysis of factors influencing observations of biased agonism at the mu-opioid receptor.

    PubMed

    Thompson, Georgina L; Lane, J Robert; Coudrat, Thomas; Sexton, Patrick M; Christopoulos, Arthur; Canals, Meritxell

    2016-08-01

    Biased agonism describes the ability of distinct G protein-coupled receptor (GPCR) ligands to stabilise distinct receptor conformations leading to the activation of different cell signalling pathways that can deliver different physiologic outcomes. This phenomenon is having a major impact on modern drug discovery as it offers the potential to design ligands that selectively activate or inhibit the signalling pathways linked to therapeutic effects with minimal activation or blockade of signalling pathways that are linked to the development of adverse on-target effects. However, the explosion in studies of biased agonism at multiple GPCR families in recombinant cell lines has revealed a high degree of variability on descriptions of biased ligands at the same GPCR and raised the question of whether biased agonism is a fixed attribute of a ligand in all cell types. The current study addresses this question at the mu-opioid receptor (MOP). Here, we have systematically assessed the impact of differential cellular protein complement (and cellular background), signalling kinetics and receptor species on our previous descriptions of biased agonism at MOP by several opioid peptides and synthetic opioids. Our results show that all these factors need to be carefully determined and reported when considering biased agonism. Nevertheless, our studies also show that, despite changes in overall signalling profiles, ligands that previously showed distinct bias profiles at MOP retained their uniqueness across different cell backgrounds. PMID:27286929

  12. Comparative modeling and molecular dynamics studies of the delta, kappa and mu opioid receptors.

    PubMed

    Strahs, D; Weinstein, H

    1997-09-01

    Molecular models of the trans-membrane domains of delta, kappa and mu opioid receptors, members of the G-protein coupled receptor (GPCR) superfamily, were developed using techniques of homology modeling and molecular dynamics simulations. Structural elements were predicted from sequence alignments of opioid and related receptors based on (i) the consensus, periodicities and biophysical interpretations of alignment-derived properties, and (ii) tertiary structure homology to rhodopsin. Initial model structures of the three receptors were refined computationally with energy minimization and the result of the first 210 ps of a 2 ns molecular dynamics trajectory at 300K. Average structures from the trajectory obtained for each receptor subtype after release of the initial backbone constraints show small backbone deviations, indicating stability. During the molecular dynamics phase, subtype-differentiated residues of the receptors developed divergent structures within the models, including changes in regions common to the three subtypes and presumed to belong to ligand binding regions. The divergent features developed by the model structures appear to be consistent with the observed ligand binding selectivities of the opioid receptors. The results thus implicate identifiable receptor microenvironments as primary determinants of some of the observed subtype specificities in opiate ligand binding and in functional effects of mutagenesis. Networks of interacting residues observed in the models are common to the opiate receptors and other GPCRs, indicating core interfaces that are potentially responsible for structural integrity and signal transduction. Analysis of extended molecular dynamics trajectories reveals concerted motions of distant parts of ligand-binding regions, suggesting motion-sensitive components of ligand binding. The comparative modeling results from this study help clarify experimental observations of subtype differences and suggest both structural and

  13. Crystal structure of the[mu]-opioid receptor bound to a morphinan antagonist

    SciTech Connect

    Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Mathiesen, Jesper M.; Sunahara, Roger K.; Pardo, Leonardo; Weis, William I.; Kobilka, Brian K.; Granier, Sébastien

    2012-06-27

    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled {mu}-opioid receptor ({mu}-OR) in the central nervous system. Here we describe the 2.8 {angstrom} crystal structure of the mouse {mu}-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the {mu}-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  14. Affinity of the enantiomers of. alpha. - and. beta. -cyclazocine for binding to the phencyclidine and. mu. opioid receptors

    SciTech Connect

    Todd, S.L.; Balster, R.L.; Martin, B.R. )

    1990-01-01

    The enantiomers in the {alpha} and {beta} series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and {mu}-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-{beta}-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-{alpha}-cyclazocine, (+)-{alpha}-cyclazocine, and (+)-{beta}-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of ({sup 3}H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent K{sub D} of 1.9 nM and an estimated Bmax of 117 pM. (3H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-{alpha}-cyclazocine (K{sub D} = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-{beta}-cyclazocine, (+)-{alpha}-cyclazocine and (+)-{beta}-cyclazocine, respectively, for binding to the {mu}-opioid receptor. These data show that, although (-)-{beta}-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptor since it also potently displaces {mu}-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.

  15. Remifentanil produces cross-desensitization and tolerance with morphine on the mu-opioid receptor.

    PubMed

    Nowoczyn, M; Marie, N; Coulbault, L; Hervault, M; Davis, A; Hanouz, J L; Allouche, S

    2013-10-01

    Remifentanil is a powerful mu-opioid (MOP) receptor agonist used in anaesthesia with a very short half-life. However, per-operative perfusion of remifentanil was shown to increase morphine consumption during post-operative period to relieve pain. In the present study, we aimed to describe the cellular mechanisms responsible for this apparent reduction of morphine efficacy. For this purpose, we first examined the pharmacological properties of both remifentanil and morphine at the MOP receptor, endogenously expressed in the human neuroblastoma SH-SY5Y cell line, to regulate adenylyl cyclase and the MAP kinase ERK1/2 pathway, their potency to promote MOP receptor phosphorylation, arrestin 3-CFP (cyan fluorescent protein) recruitment and receptor trafficking during acute and sustained exposure. In the second part of this work, we studied the effects of a prior exposure of remifentanil on morphine-induced inhibition of cAMP accumulation, activation of ERK1/2 and analgesia. We showed that sustained exposure to remifentanil promoted a rapid desensitization of opioid receptors on both signalling pathways and a pretreatment with this agonist reduced signal transduction produced by a second challenge with morphine. While both opioid agonists promoted Ser(375) phosphorylation on MOP receptor, remifentanil induced a rapid internalization of opioid receptors compared to morphine but without detectable arrestin 3-CFP translocation to the plasma membrane in our experimental conditions. Lastly, a cross-tolerance between remifentanil and morphine was observed in mice using the hot plate test. Our in vitro and in vivo data thus demonstrated that remifentanil produced a rapid desensitization and internalization of the MOP receptor that would reduce the anti-nociceptive effects of morphine. PMID:23792280

  16. Mu Opioid Receptor Binding Correlates with Nicotine Dependence and Reward in Smokers

    PubMed Central

    Brasic, James R.; Contoreggi, Carlo; Cascella, Nicola; Mackowick, Kristen M.; Taylor, Richard; Rousset, Olivier; Willis, William; Huestis, Marilyn A.; Concheiro, Marta; Wand, Gary; Wong, Dean F.; Volkow, Nora D.

    2014-01-01

    The rewarding effects of nicotine are associated with activation of nicotine receptors. However, there is increasing evidence that the endogenous opioid system is involved in nicotine's rewarding effects. We employed PET imaging with [11C]carfentanil to test the hypotheses that acute cigarette smoking increases release of endogenous opioids in the human brain and that smokers have an upregulation of mu opioid receptors (MORs) when compared to nonsmokers. We found no significant changes in binding potential (BPND) of [11C]carfentanil between the placebo and the active cigarette sessions, nor did we observe differences in MOR binding between smokers and nonsmokers. Interestingly, we showed that in smokers MOR availability in bilateral superior temporal cortices during the placebo condition was negatively correlated with scores on the Fagerström Test for Nicotine Dependence (FTND). Also in smokers, smoking-induced decreases in [11C]carfentanil binding in frontal cortical regions were associated with self-reports of cigarette liking and wanting. Although we did not show differences between smokers and nonsmokers, the negative correlation with FTND corroborates the role of MORs in superior temporal cortices in nicotine addiction and provides preliminary evidence of a role of endogenous opioid signaling in frontal cortex in nicotine reward. PMID:25493427

  17. Partial agonistic effect of 9-hydroxycorynantheidine on mu-opioid receptor in the guinea-pig ileum.

    PubMed

    Matsumoto, Kenjiro; Takayama, Hiromitsu; Ishikawa, Hayato; Aimi, Norio; Ponglux, Dhavadee; Watanabe, Kazuo; Horie, Syunji

    2006-04-01

    Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that is reported to have opioid agonistic properties. The 9-demethyl analogue of mitragynine, 9-hydroxycorynantheidine, is synthesized from mitragynine. 9-Hydroxycorynantheidine inhibited electrically stimulated guinea-pig ileum contraction, but its maximum inhibition was weaker than that of mitragynine and its effect was antagonized by naloxone, suggesting that 9-hydroxycorynantheidine possesses partial agonist properties on opioid receptors. Receptor binding assays revealed that 9-hydroxycorynantheidine has high affinity for mu-opioid receptors. In an assay of the guinea-pig ileum, naloxone shifted the concentration-response curves for [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), (5alpha,7alpha,8beta)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) and 9-hydroxycorynantheidine to the right in a competitive manner. The pA(2) values of naloxone against 9-hydroxycorynantheidine and DAMGO were very similar, but not that against U69593. As indicated by the two assay systems, the opioid effect of 9-hydroxycorynantheidine is selective for the mu-opioid receptor. 9-Hydroxycorynantheidine shifted the concentration-response curve for DAMGO slightly to the right. Pretreatment with the mu-opioid selective and irreversible antagonist beta-funaltorexamine hydrochloride (beta-FNA) shifted the concentration-response curve for DAMGO to the right without affecting the maximum response. On the other hand, beta-FNA did not affect the curve for 9-hydroxycorynantheidine, but decreased the maximum response because of the lack of spare receptors. These studies suggest that 9-hydroxycorynantheidine has partial agonist properties on mu-opioid receptors in the guinea-pig ileum. PMID:16266723

  18. A topographical model of mu-opioid and brain somatostatin receptor selective ligands. NMR and molecular dynamics studies.

    PubMed

    Kazmierski, W M; Ferguson, R D; Lipkowski, A W; Hruby, V J

    1995-01-01

    We have refined the 1H NMR-based conformations of the mu-opioid receptor selective peptides related to somatostatin of general formula Xxx-Yyy1-Cys-Zzz-D-Trp-Lys(Orn)5-Thr-Pen-Thr8- NH2, where Xxx, Yyy, Zzz are 0, D-Phe and Tyr for 1; 0, D-Tic and Tyr for 2; Gly, D-Tic and Tyr for 3; and 0, D-Phe and Tic for 4, respectively, (Kazmierski et al., J. Am. Chem. 113, 2275-2283), using a molecular-dynamics approach. We present evidence that the NMR data are compatible with beta II'-, gamma- and gamma'-turns for the central tetrapeptide Tyr-D-Trp-Lys/Orn-Thr. Based on detailed structural and topographical considerations, we suggest that the mu-opioid receptor selectivity of 2 is due to a particular spatial arrangement of aromatic side chains of D-Tic1 and Tyr3 (7.5 A), and that the opioid receptor recognition domain is located in the N-terminal part of the peptide while the somatostatin receptor recognition domain is determined by the central, turn forming part of this class of cyclic peptides. A model for a mu-opioid selective ligand has emerged from these studies that shows excellent structural similarities to rigid opioid alkaloids. PMID:8537180

  19. Graphene decorated with mu-opioid receptor: the ionic screening effect and detection of enkephalin

    NASA Astrophysics Data System (ADS)

    Ping, Jinglei; Johnson, A. T. Charlie; Liu, Renyu; A. T. Charlie Johnson Team; Renyu Liu Collaboration

    2015-03-01

    We investigated the properties of graphene field effect transistors (GFETs) decorated with a computaionally redesigned, water-soluble variant of the human mu-opioid receptor (wsMOR) in physiological buffer solution. The shift of the Fermi level in the GFETs is quantitatively described by chemical-gating effect of charges on the wsMOR that are screened by the ionic solution. Our results suggest that sensitivity to the molecular target is lost when the Debye screening length of the solution is shorter than the distance from the graphene to the wsMOR; thus de-salting may be necessary when wsMOR decorated GFETs are used as biosensors in solution. We used this insight to detect DAMGO, a synthetic analog to the endogenous opioid peptide encephalin, at a concentration of 10 pM (5.1 pg/mL) in artificial cerebrospinal fluid (aCSF) diluted to 5% of its normal salt concentration. When the sensors were measured in a dry state, the limit of detection for DAGMO was 1 pM (0.5 pg/mL), one-third of the baseline in human body.Funding for this work was provided by DARPA.

  20. Involvement of mu-opioid receptors in antinociception and inhibition of gastrointestinal transit induced by 7-hydroxymitragynine, isolated from Thai herbal medicine Mitragyna speciosa.

    PubMed

    Matsumoto, Kenjiro; Hatori, Yoshio; Murayama, Toshihiko; Tashima, Kimihito; Wongseripipatana, Sumphan; Misawa, Kaori; Kitajima, Mariko; Takayama, Hiromitsu; Horie, Syunji

    2006-11-01

    7-hydroxymitragynine, a constituent of the Thai herbal medicine Mitragyna speciosa, has been found to have a potent opioid antinociceptive effect. In the present study, we investigated the mechanism of antinociception and the inhibitory effect on gastrointestinal transit of 7-hydroxymitragynine, and compared its effects with those of morphine. When administered subcutaneously to mice, 7-hydroxymitragynine produced antinociceptive effects about 5.7 and 4.4 times more potent than those of morphine in the tail-flick (ED50=0.80 mg/kg) and hot-plate (ED50=0.93 mg/kg) tests, respectively. The antinociceptive effect of 7-hydroxymitragynine was significantly blocked by the mu1/mu2-opioid receptor antagonist beta-funaltrexamine hydrochloride (beta-FNA) and the mu1-opioid receptor-selective antagonist naloxonazine in both tests. Thus, 7-hydroxymitragynine acts predominantly on mu-opioid receptors, especially on mu1-opioid receptors. Isolated tissue studies further supported its specificity for the mu-opioid receptors. Further, 7-hydroxymintragynine dose-dependently (ED50=1.19 mg/kg, s.c.) and significantly inhibited gastrointestinal transit in mice, as morphine does. The inhibitory effect was significantly antagonized by beta-FNA pretreatment, but slightly antagonized by naloxonazine. The ED50 value of 7-hydroxymitragynine on gastrointestinal transit was larger than its antinociceptive ED50 value. On the other hand, morphine significantly inhibits gastrointestinal transit at a much smaller dose than its antinociceptive dose. These results suggest that mu-opioid receptor mechanisms mediate the antinociceptive effect and inhibition of gastrointestinal transit. This compound induced more potent antinociceptive effects and was less constipating than morphine. PMID:16978601

  1. Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target

    PubMed Central

    Morse, Megan; Tran, Elizabeth; Sun, Haiyan; Levenson, Robert; Fang, Ye

    2011-01-01

    Development of new opioid drugs that provide analgesia without producing dependence is important for pain treatment. Opioid agonist drugs exert their analgesia effects primarily by acting at the mu opioid receptor (MOR) sites. High-resolution differentiation of opioid ligands is crucial for the development of new lead drug candidates with better tolerance profiles. Here, we use a label-free integrative pharmacology on-target (iPOT) approach to characterize the functional selectivity of a library of known opioid ligands for the MOR. This approach is based on the ability to detect dynamic mass redistribution (DMR) arising from the activation of the MOR in living cells. DMR assays were performed in HEK-MOR cells with and without preconditioning with probe molecules using label-free resonant waveguide grating biosensors, wherein the probe molecules were used to modify the activity of specific signaling proteins downstream the MOR. DMR signals obtained were then translated into high resolution heat maps using similarity analysis based on a numerical matrix of DMR parameters. Our data indicate that the iPOT approach clearly differentiates functional selectivity for distinct MOR signaling pathways among different opioid ligands, thus opening new avenues to discover and quantify the functional selectivity of currently used and novel opioid receptor drugs. PMID:22003401

  2. Mu opioid receptor localization in the basolateral amygdala: An ultrastructural analysis.

    PubMed

    Zhang, J; Muller, J F; McDonald, A J

    2015-09-10

    Receptor binding studies have shown that the density of mu opioid receptors (MORs) in the basolateral amygdala is among the highest in the brain. Activation of these receptors in the basolateral amygdala is critical for stress-induced analgesia, memory consolidation of aversive events, and stress adaptation. Despite the importance of MORs in these stress-related functions, little is known about the neural circuits that are modulated by amygdalar MORs. In the present investigation light and electron microscopy combined with immunohistochemistry was used to study the expression of MORs in the anterior basolateral nucleus (BLa). At the light microscopic level, light to moderate MOR-immunoreactivity (MOR-ir) was observed in a small number of cell bodies of nonpyramidal interneurons and in a small number of processes and puncta in the neuropil. At the electron microscopic level most MOR-ir was observed in dendritic shafts, dendritic spines, and axon terminals. MOR-ir was also observed in the Golgi apparatus of the cell bodies of pyramidal neurons (PNs) and interneurons. Some of the MOR-positive (MOR+) dendrites were spiny, suggesting that they belonged to PNs, while others received multiple asymmetrical synapses typical of interneurons. The great majority of MOR+ axon terminals (80%) that formed synapses made asymmetrical (excitatory) synapses; their main targets were spines, including some that were MOR+. The main targets of symmetrical (inhibitory and/or neuromodulatory) synapses were dendritic shafts, many of which were MOR+, but some of these terminals formed synapses with somata or spines. All of our observations were consistent with the few electrophysiological studies which have been performed on MOR activation in the basolateral amygdala. Collectively, these findings suggest that MORs may be important for filtering out weak excitatory inputs to PNs, allowing only strong inputs or synchronous inputs to influence pyramidal neuronal firing. PMID:26164501

  3. Agonist-selective mechanisms of mu-opioid receptor desensitization in human embryonic kidney 293 cells.

    PubMed

    Johnson, Elizabeth A; Oldfield, Sue; Braksator, Ellen; Gonzalez-Cuello, Ana; Couch, Daniel; Hall, Kellie J; Mundell, Stuart J; Bailey, Chris P; Kelly, Eamonn; Henderson, Graeme

    2006-08-01

    The ability of two opioid agonists, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and morphine, to induce mu-opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different

  4. Variation at the mu-opioid receptor gene (OPRM1) influences attachment behavior in infant primates.

    PubMed

    Barr, Christina S; Schwandt, Melanie L; Lindell, Stephen G; Higley, J Dee; Maestripieri, Dario; Goldman, David; Suomi, Stephen J; Heilig, Markus

    2008-04-01

    In a variety of species, development of attachment to a caregiver is crucial for infant survival and partly mediated by the endogenous opioids. Functional mu-opioid receptor gene polymorphisms are present in humans (OPRM1 A118G) and rhesus macaques (OPRM1 C77G). We hypothesized that rhesus infants carrying a gain-of-function OPRM1 77G allele would experience increased reward during maternal contact and would, therefore, display increased measures of attachment. We collected behavioral data from rhesus macaques (n = 97) during early infancy and at 6 months of age, across four cycles of maternal separation (4 days) and reunion (3 days). Animals were genotyped for the OPRM1 C77G polymorphism, and the effects of this allele on attachment-related behaviors were analyzed. Infants carrying the G allele exhibited higher levels of attachment behavior during early infancy. During prolonged periods of maternal separation, although infant macaques homozygous for the C allele exhibited decreases in their levels of distress vocalization with repeated separation, this response persisted in G allele carriers. The OPRM1 77G allele also affected social preference during reunion. C/G infants spent increasing amounts of time in social contact with their mothers as a function of repeated separation and were less likely to interact with other individuals in the social group, a pattern not observed among infants with the C/C genotype. These findings suggest a role for OPRM1 variation in the expression of attachment behavior in human subjects, especially as a function of separation from the caregiver. PMID:18378897

  5. Epigenetic Variation in the Mu-opioid Receptor Gene in Infants with Neonatal Abstinence Syndrome

    PubMed Central

    Wachman, Elisha M; Hayes, Marie J; Lester, Barry M; Terrin, Norma; Brown, Mark S; Nielsen, David A; Davis, Jonathan M

    2014-01-01

    Objective Neonatal abstinence syndrome (NAS) from in utero opioid exposure is highly variable with genetic factors appearing to play an important role. Epigenetic changes in cytosine:guanine (CpG) dinucleotide methylation can occur after drug exposure and may help to explain NAS variability. We correlated DNA methylation levels in the mu-opioid receptor (OPRM1) promoter in opioid-exposed infants and correlate them with NAS outcomes. Study design DNA samples from cord blood or saliva were analyzed for 86 infants being treated for NAS according to institutional protocol. Methylation levels at 16 OPRM1 CpG sites were determined and correlated with NAS outcome measures, including need for treatment, treatment with >2 medications, and length of hospital stay. We adjusted for co-variates and multiple genetic testing. Results Sixty-five percent of infants required treatment for NAS, and 24% required ≥2 medications. Hypermethylation of the OPRM1 promoter was measured at the −10 CpG in treated versus non-treated infants [adjusted difference δ=3.2% (95% CI 0.3–6.0%), p=0.03; NS after multiple testing correction]. There was hypermethylation at the −14 [δ=4.9% (95% CI 1.8–8.1%), p=0.003], −10 [δ=5.0% (95% CI 2.3–7.7%), p=0.0005)], and +84 [δ=3.5% (95% CI 0.6 – 6.4), p=0.02] CpG sites in infants requiring ≥2 medications which remained significant for −14 and −10 after multiple testing correction. Conclusions Increased methylation within the OPRM1 promoter is associated with worse NAS outcomes, consistent with gene silencing. PMID:24996986

  6. Affinity, potency and efficacy of tramadol and its metabolites at the cloned human mu-opioid receptor.

    PubMed

    Gillen, C; Haurand, M; Kobelt, D J; Wnendt, S

    2000-08-01

    The present study was conducted to characterise the centrally active analgesic drug tramadol hydrochloride [(1RS,2RS)-2-[(dimethyl-amino)-methyl]-1-(3-methoxyphenyl)-cyclohe xanol hydrochloride] and its metabolites M1, M2, M3, M4 and M5 at the cloned human mu-opioid receptor. Membranes from stably transfected Chinese hamster ovary (CHO) cells were used to determine the four parameters of the ligand-receptor interaction: the affinity of (+/-)-tramadol and its metabolites was determined by competitive inhibition of [3H]naloxone binding under high and low salt conditions. The agonist-induced stimulation of [35S]GTPgammaS binding permits the measurement of potency (EC50), efficacy (Emax = maximal stimulation) and relative intrinsic efficacy (effect as a function of receptor occupation). The metabolite (+)-M1 showed the highest affinity (Ki=3.4 nM) to the human mu-opioid receptor, followed by (+/-)-M5 (Ki=100 nM), (-)-M1 (Ki=240 nM) and (+/-)-tramadol (Ki=2.4 microM). The [35S]GTPgammaS binding assay revealed an agonistic activity for the metabolites (+)-M1, (-)-M1 and (+/-)-M5 with the following rank order of intrinsic efficacy: (+)-M1>(+/-)-M5>(-)-M1. The metabolites (+/-)-M2, (+/-)-M3 and (+/-)-M4 displayed only weak affinity (Ki> 10 microM) and had no stimulatory effect on GTPgammaS binding. These data indicate that the metabolite (+)-M1 is responsible for the mu-opioid-derived analgesic effect. PMID:10961373

  7. Mu Opioid Receptor Modulation of Dopamine Neurons in the Periaqueductal Gray/Dorsal Raphe: A Role in Regulation of Pain.

    PubMed

    Li, Chia; Sugam, Jonathan A; Lowery-Gionta, Emily G; McElligott, Zoe A; McCall, Nora M; Lopez, Alberto J; McKlveen, Jessica M; Pleil, Kristen E; Kash, Thomas L

    2016-07-01

    The periaqueductal gray (PAG) is a brain region involved in nociception modulation, and an important relay center for the descending nociceptive pathway through the rostral ventral lateral medulla. Given the dense expression of mu opioid receptors and the role of dopamine in pain, the recently characterized dopamine neurons in the ventral PAG (vPAG)/dorsal raphe (DR) region are a potentially critical site for the antinociceptive actions of opioids. The objectives of this study were to (1) evaluate synaptic modulation of the vPAG/DR dopamine neurons by mu opioid receptors and to (2) dissect the anatomy and neurochemistry of these neurons, in order to assess the downstream loci and functions of their activation. Using a mouse line that expresses eGFP under control of the tyrosine hydroxylase (TH) promoter, we found that mu opioid receptor activation led to a decrease in inhibitory inputs onto the vPAG/DR dopamine neurons. Furthermore, combining immunohistochemistry, optogenetics, electrophysiology, and fast-scan cyclic voltammetry in a TH-cre mouse line, we demonstrated that these neurons also express the vesicular glutamate type 2 transporter and co-release dopamine and glutamate in a major downstream projection structure-the bed nucleus of the stria terminalis. Finally, activation of TH-positive neurons in the vPAG/DR using Gq designer receptors exclusively activated by designer drugs displayed a supraspinal, but not spinal, antinociceptive effect. These results indicate that vPAG/DR dopamine neurons likely play a key role in opiate antinociception, potentially via the activation of downstream structures through dopamine and glutamate release. PMID:26792442

  8. Mu opioid receptor modulation in the nucleus accumbens lowers voluntary wheel running in rats bred for high running motivation.

    PubMed

    Ruegsegger, Gregory N; Toedebusch, Ryan G; Will, Matthew J; Booth, Frank W

    2015-10-01

    The exact role of opioid receptor signaling in mediating voluntary wheel running is unclear. To provide additional understanding, female rats selectively bred for motivation of low (LVR) versus high voluntary running (HVR) behaviors were used. Aims of this study were 1) to identify intrinsic differences in nucleus accumbens (NAc) mRNA expression of opioid-related transcripts and 2) to determine if nightly wheel running is differently influenced by bilateral NAc injections of either the mu-opioid receptor agonist D-Ala2, NMe-Phe4, Glyo5-enkephalin (DAMGO) (0.25, 2.5 μg/side), or its antagonist, naltrexone (5, 10, 20 μg/side). In Experiment 1, intrinsic expression of Oprm1 and Pdyn mRNAs were higher in HVR compared to LVR. Thus, the data imply that line differences in opioidergic mRNA in the NAc could partially contribute to differences in wheel running behavior. In Experiment 2, a significant decrease in running distance was present in HVR rats treated with 2.5 μg DAMGO, or with 10 μg and 20 μg naltrexone between hours 0-1 of the dark cycle. Neither DAMGO nor naltrexone had a significant effect on running distance in LVR rats. Taken together, the data suggest that the high nightly voluntary running distance expressed by HVR rats is mediated by increased endogenous mu-opioid receptor signaling in the NAc, that is disturbed by either agonism or antagonism. In summary, our findings on NAc opioidergic mRNA expression and mu-opioid receptor modulations suggest HVR rats, compared to LVR rats, express higher running levels mediated by an increase in motivation driven, in part, by elevated NAc opioidergic signaling. PMID:26044640

  9. Functional mu opioid receptors are expressed in cholinergic interneurons of the rat dorsal striatum: territorial specificity and diurnal variation.

    PubMed

    Jabourian, Maritza; Venance, Laurent; Bourgoin, Sylvie; Ozon, Sylvie; Pérez, Sylvie; Godeheu, Gérard; Glowinski, Jacques; Kemel, Marie-Louise

    2005-06-01

    Striatal cholinergic interneurons play a crucial role in the control of movement as well as in motivational and learning aspects of behaviour. Neuropeptides regulate striatal cholinergic transmission and particularly activation of mu opioid receptor (MOR) inhibits acetylcholine (ACh) release in the dorsal striatum. In the present study we investigated whether this cholinergic transmission could be modulated by an enkephalin/MOR direct process. We show that mRNA and protein of MORs are expressed by cholinergic interneurons in the limbic/prefrontal territory but not by those in the sensorimotor territory of the dorsal striatum. These MORs are functional because potassium-evoked release of ACh from striatal synaptosomes was dose-dependently reduced by a selective MOR agonist, this effect being suppressed by a MOR antagonist. The MOR regulation of cholinergic interneurons presented a diurnal variation. (i) The percentage of cholinergic interneurons containing MORs that was 32% at the beginning of the light period (morning) increased to 80% in the afternoon. (ii) The MOR-mediated inhibition of synaptosomal ACh release was higher in the afternoon than in the morning. (iii) While preproenkephalin mRNA levels remained stable, enkephalin tissue content was the lowest (-32%) in the afternoon when the spontaneous (+35%) and the N-methyl-d-aspartate-evoked (+140%) releases of enkephalin (from microsuperfused slices) were the highest. Therefore, by acting on MORs present on cholinergic interneurons, endogenously released enkephalin reduces ACh release. This direct enkephalin/MOR regulation of cholinergic transmission that operates only in the limbic/prefrontal territory of the dorsal striatum might contribute to information processing in fronto-cortico-basal ganglia circuits. PMID:16026468

  10. Endometriosis Is Associated With a Shift in MU Opioid and NMDA Receptor Expression in the Brain Periaqueductal Gray.

    PubMed

    Torres-Reverón, Annelyn; Palermo, Karylane; Hernández-López, Anixa; Hernández, Siomara; Cruz, Myrella L; Thompson, Kenira J; Flores, Idhaliz; Appleyard, Caroline B

    2016-09-01

    Studies have examined how endometriosis interacts with the nervous system, but little attention has been paid to opioidergic systems, which are relevant to pain signaling. We used the autotransplantation rat model of endometriosis and allowed to progress for 60 days. The brain was collected and examined for changes in endogenous opioid peptides, mu opioid receptors (MORs), and the N-methyl-d-aspartate subunit receptor (NR1) in the periaqueductal gray (PAG), since both of these receptors can regulate PAG activity. No changes in endogenous opioid peptides in met- and leu-enkephalin or β-endorphin levels were observed within the PAG. However, MOR immunoreactivity was significantly decreased in the ventral PAG in the endometriosis group. Endometriosis reduced by 20% the number of neuronal profiles expressing MOR and reduced by 40% the NR1 profiles. Our results suggest that endometriosis is associated with subtle variations in opioidergic and glutamatergic activity within the PAG, which may have implications for pain processing. PMID:27089914

  11. Neurokinin1 receptors regulate morphine-induced endocytosis and desensitization of mu opioid receptors in CNS neurons

    PubMed Central

    Yu, Y. Joy; Arttamangkul, Seksiri; Evans, Christopher J.; Williams, John T.; von Zastrow, Mark

    2009-01-01

    Mu opioid receptors (MORs) are G protein-coupled receptors (GPCRs) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are co-expressed with neurokinin 1 receptors (NK1Rs) in several regions of the central nervous system (CNS) that control opioid dependence and reward. NK1R activation affects opioid reward specifically, however, and the cellular basis for this specificity is unknown. We found that ligand-induced activation of NK1Rs produces a cell autonomous and non-reciprocal inhibition of MOR endocytosis induced by diverse opioids. Studies using epitope-tagged receptors expressed in cultured striatal neurons and a neuroblastoma cell model indicated that this heterologous regulation is mediated by NK1R-dependent sequestration of arrestins on endosome membranes. First, endocytic inhibition mediated by wild type NK1Rs was overcome in cells over-expressing β-arrestin2, a major arrestin isoform expressed in striatum. Second, NK1R activation promoted sequestration of β-arrestin2 on endosomes, whereas MOR activation did not. Third, heterologous inhibition of MOR endocytosis was prevented by mutational disruption of β-arrestin2 sequestration by NK1Rs. NK1R-mediated regulation of MOR trafficking was associated with reduced opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Further, heterologous regulation of MOR trafficking was observed in both amygdala and locus coeruleus neurons that naturally co-express these receptors. These results identify a cell autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons. PMID:19129399

  12. Design, Syntheses, and Biological Evaluation of 14-Heteroaromatic Substituted Naltrexone Derivatives: Pharmacological Profile Switch from Mu Opioid Receptor Selectivity to Mu/Kappa Opioid Receptor Dual Selectivity

    PubMed Central

    Yuan, Yunyun; Zaidi, Saheem A.; Elbegdorj, Orgil; Aschenbach, Lindsey C. K.; Li, Guo; Stevens, David L.; Scoggins, Krista L.; Dewey, William L.; Selley, Dana E.; Zhang, Yan

    2015-01-01

    Based on a mu opioid receptor (MOR) homology model and the “isosterism” concept, three generations of 14-heteroaromatically substituted naltrexone derivatives were designed, synthesized, and evaluated as potential MOR selective ligands. The first generation ligands appeared to be MOR selective, whereas the second and the third generation ones showed MOR/kappa opioid receptor (KOR) dual selectivity. Docking of ligands 2 (MOR selective) and 10 (MOR/KOR dual selective) to the three opioid receptor crystal structures revealed a non-conserved residue facilitated “hydrogen bonding network” that could be responsible for their distinctive selectivity profiles. The MOR/KOR dual selective ligand 10 showed no agonism and acted as a potent antagonist in the tail flick assay. It also produced less severe opioid withdrawal symptoms than naloxone in morphine dependent mice. In conclusion, ligand 10 may serve as a novel lead compound to develop MOR/KOR dual selective ligands, which might possess unique therapeutic value for opioid addiction treatment. PMID:24144240

  13. Mu-opioid receptor activation in the medial shell of nucleus accumbens promotes alcohol consumption, self-administration and cue-induced reinstatement.

    PubMed

    Richard, Jocelyn M; Fields, Howard L

    2016-09-01

    Endogenous opioid signaling in ventral cortico-striatal-pallidal circuitry is implicated in elevated alcohol consumption and relapse to alcohol seeking. Mu-opioid receptor activation in the medial shell of the nucleus accumbens (NAc), a region implicated in multiple aspects of reward processing, elevates alcohol consumption while NAc opioid antagonists reduce it. However, the precise nature of the increases in alcohol consumption, and the effects of mu-opioid agonists on alcohol seeking and relapse are not clear. Here, we tested the effects of the mu-opioid agonist [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin (DAMGO) in rat NAc shell on lick microstructure in a free-drinking test, alcohol seeking during operant self-administration, extinction learning and expression, and cue-reinforced reinstatement of alcohol seeking. DAMGO enhanced the number, but not the size of drinking bouts. DAMGO also enhanced operant alcohol self-administration and cue-induced reinstatement, but did not affect extinction learning or elicit reinstatement in the absence of cues. Our results suggest that mu-opioid agonism in NAc shell elevates alcohol consumption, seeking and conditioned reinforcement primarily by enhancing the incentive motivational properties of alcohol and alcohol-paired cues, rather than by modulating palatability, satiety, or reinforcement. PMID:27089981

  14. Preprodynorphin mediates locomotion and D2 dopamine and mu-opioid receptor changes induced by chronic 'binge' cocaine administration.

    PubMed

    Bailey, A; Yoo, J H; Racz, I; Zimmer, A; Kitchen, I

    2007-09-01

    Evidence suggests that the kappa-opioid receptor (KOP-r) system plays an important role in cocaine addiction. Indeed, cocaine induces endogenous KOP activity, which is a mechanism that opposes alterations in behaviour and brain function resulting from repeated cocaine use. In this study, we have examined the influence of deletion of preprodynorphin (ppDYN) on cocaine-induced behavioural effects and on hypothalamic-pituitary-adrenal axis activity. Furthermore, we have measured mu-opioid receptor (MOP-r) agonist-stimulated [(35)S]GTPgammaS, dopamine D(1), D(2) receptor and dopamine transporter (DAT) binding. Male wild-type (WT) and ppDYN knockout (KO) mice were injected with saline or cocaine (45 mg/kg/day) in a 'binge' administration paradigm for 14 days. Chronic cocaine produced an enhancement of locomotor sensitisation in KO. No genotype effect was found on stereotypy behaviour. Cocaine-enhanced MOP-r activation in WT but not in KO. There was an overall decrease in D(2) receptor binding in cocaine-treated KO but not in WT mice. No changes were observed in D(1) and DAT binding. Cocaine increased plasma corticosterone levels in WT but not in KO. The data confirms that the endogenous KOP system inhibits dopamine neurotransmission and that ppDYN may mediate the enhancement of MOP-r activity and the activation of the hypothalamic-pituitary-adrenal axis after chronic cocaine treatment. PMID:17532787

  15. Abolished thermal and mechanical antinociception but retained visceral chemical antinociception induced by butorphanol in mu-opioid receptor knockout mice.

    PubMed

    Ide, Soichiro; Minami, Masabumi; Ishihara, Kumatoshi; Uhl, George R; Satoh, Masamichi; Sora, Ichiro; Ikeda, Kazutaka

    2008-06-01

    Butorphanol is hypothesized to induce analgesia via opioid pathways, although the precise mechanisms for its effects remain unknown. In this study, we investigated the role of the mu-opioid receptor (MOP) in thermal, mechanical, and visceral chemical antinociception induced by butorphanol using MOP knockout (KO) mice. Butorphanol-induced thermal antinociception, assessed by the hot-plate and tail-flick tests, was significantly reduced in heterozygous and abolished in homozygous MOP-KO mice compared with wildtype mice. The results obtained from our butorphanol-induced mechanical antinociception experiments, assessed by the Randall-Selitto test, were similar to the results obtained from the thermal antinociception experiments in these mice. Interestingly, however, butorphanol retained its ability to induce significant visceral chemical antinociception, assessed by the writhing test, in homozygous MOP-KO mice. The butorphanol-induced visceral chemical antinociception that was retained in homozygous MOP-KO mice was completely blocked by pretreatment with nor-binaltorphimine, a kappa-opioid receptor (KOP) antagonist. In vitro binding and cyclic adenosine monophosphate assays also showed that butorphanol possessed higher affinity for KOPs and MOPs than for delta-opioid receptors. These results molecular pharmacologically confirmed previous studies implicating MOPs, and partially KOPs, in mediating butorphanol-induced analgesia. PMID:18417173

  16. Reconstitution of rate brain /mu/ opioid receptors with purified guanine nucleotide-binding regulatory proteins, G/sub i/ and G/sub o/

    SciTech Connect

    Ueda, Hiroshi; Harada, Hitoshi; Nozaki, Masakatsu; Katada, Toshiaki; Ui, Michio; Satoh, Masamichi; Takagi, Hiroshi

    1988-09-01

    Reconstitution of purified /mu/ opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. The purified /mu/ opioid receptor (pI 5.6) migrated as a single M/sub r/ 58,000 polypeptide by NaDodSO/sub 4//PAGE, a value identical to that obtained by affinity cross-linking purified /mu/ receptors. When purified /mu/ receptors were reconstituted with purified G/sub i/, the G protein that mediates the inhibition of adenylate cyclase, the displacement of (/sup 3/H)naloxone binding by (D-Ala/sup 2/,MePhe/sup 4/,Gly-ol/sup 5/)enkephalin was increased 215-fold; this increase was abolished by adding 100 /mu/M guanosine 5'-(/gamma/-thio)triphosphate. Similar increases in agonist displacement of (/sup 3/H)naloxone binding (33-fold) and its abolition by guanosine 5'-(/gamma/-thio)triphosphate were observed with G/sub o/, the G protein of unknown function, but not with the v-Ki-ras protein p.21. The stoichiometry was such that the stimulation of 1 mol of /mu/ receptor led to the binding of (/sup 3/H)guanosine 5'-(/beta/,/gamma/-imido)triphosphate to 2.5 mol of G/sub i/ or to 1.37 mol of G/sub o/. These results suggest that the purified /mu/ opioid receptor is functionally coupled to G/sub i/ and G/sub o/ in the reconstituted phospholipid vesicles.

  17. Morphine-induced antinociception and reward in "humanized" mice expressing the mu opioid receptor A118G polymorphism.

    PubMed

    Henderson-Redmond, Angela N; Yuill, Matthew B; Lowe, Tammy E; Kline, Aaron M; Zee, Michael L; Guindon, Josée; Morgan, Daniel J

    2016-05-01

    The rewarding and antinociceptive effects of opioids are mediated through the mu-opioid receptor. The A118G single nucleotide polymorphism in this receptor has been implicated in drug addiction and differences in pain response. Clinical and preclinical studies have found that the G allele is associated with increased heroin reward and self-administration, elevated post-operative pain, and reduced analgesic responsiveness to opioids. Male and female mice homozygous for the "humanized" 118AA or 118GG alleles were evaluated to test the hypothesis that 118GG mice are less sensitive to the rewarding and antinociceptive effects of morphine. We found that 118AA and 118GG mice of both genders developed conditioned place preference for morphine. All mice developed tolerance to the antinociceptive and hypothermic effects of morphine. However, morphine tolerance was not different between AA and GG mice. We also examined sensitivity to the antinociceptive and hypothermic effects of cumulative morphine doses. We found that 118GG mice show reduced hypothermic and antinociceptive responses on the hotplate for 10mg/kg morphine. Finally, we examined basal pain response and morphine-induced antinociception in the formalin test for inflammatory pain. We found no gender or genotype differences in either basal pain response or morphine-induced antinociception in the formalin test. Our data suggests that homozygous expression of the GG allele in mice blunts morphine-induced hypothermia and hotplate antinociception but does not alter morphine CPP, morphine tolerance, or basal inflammatory pain response. PMID:26521067

  18. Estrogen facilitates and the kappa and mu opioid receptors mediate antinociception produced by intrathecal (-)-pentazocine in female rats.

    PubMed

    Robinson, Douglas L; Nag, Subodh; Mokha, Sukhbir S

    2016-10-01

    Pentazocine, a mixed-action kappa opioid receptor (KOR) agonist, has high affinity for both KOR and the mu opioid receptor (MOR), and has been shown clinically to alleviate pain with a pronounced effect in women. However, whether local application of pentazocine in the spinal cord produces antinociception and the contribution of spinal KOR and MOR in mediating the effect of pentazocine in female rats remain unknown. Also, it is not known whether pentazocine-induced antinociception in females is estrogen-dependent. Hence, we investigated whether intrathecal (i.t.) (-)-pentazocine produces thermal antinociception and whether estrogen modulates the drug effect in female rats. Only the highest dose of pentazocine (500 nmol) was effective in producing antinociception in ovariectomized (OVX) rats. In contrast, pentazocine produced antinociception in estradiol-treated ovariectomized females (OVX+E) rats with the lowest effective dose being 250nmol. KOR or MOR mediated the effect of the lowest effective dose in OVX+E rats; however, MOR blockade extended the KOR-mediated effect of 500nmol pentazocine in both groups. In normally cycling females, the 250nmol dose was effective in producing antinociception at the proestrous, but not at the diestrous stage of the estrous cycle. Thus, estrogen facilitates and KOR or MOR mediates. the antinociceptive effect of i.t. (-)-pentazocine in female rats. Selective doses of (-)-pentazocine, with or without MOR blockade, may have a therapeutic benefit. PMID:27312267

  19. mu Opioid receptor-mediated G-protein activation by heroin metabolites: evidence for greater efficacy of 6-monoacetylmorphine compared with morphine.

    PubMed

    Selley, D E; Cao, C C; Sexton, T; Schwegel, J A; Martin, T J; Childers, S R

    2001-08-15

    The efficacy of heroin metabolites for the stimulation of mu opioid receptor-mediated G-protein activation was investigated using agonist-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate binding. In rat thalamic membranes, heroin and its primary metabolite, 6-monoacetylmorphine (6-MAM), were more efficacious than morphine or morphine-6-beta D-glucuronide. This increased efficacy was not due to increased action of heroin and 6-MAM at delta receptors, as determined by competitive antagonism by naloxone, lack of antagonism by naltrindole, and competitive partial antagonism with morphine. In agreement with this interpretation, the same relative efficacy profile of heroin and its metabolites was observed at the cloned human mu opioid receptor expressed in C6 glioma cells. Moreover, these efficacy differences were GDP-dependent in a manner consistent with accepted mechanisms of receptor-mediated G-protein activation. The activity of heroin was attributed to in vitro deacetylation to 6-MAM, as confirmed by HPLC analysis. These results indicate that the heroin metabolite 6-MAM possesses higher efficacy than other heroin metabolites at mu opioid receptors, which may contribute to the higher efficacy of heroin compared with morphine in certain behavioral paradigms in vivo. PMID:11448454

  20. Acute "binge" cocaine increases mu-opioid receptor mRNA levels in areas of the rat mesolimbic mesocortical dopamine system.

    PubMed

    Yuferov, V; Zhou, Y; Spangler, R; Maggos, C E; Ho, A; Kreek, M J

    1999-01-01

    Autoradiography studies demonstrated that chronic "binge" cocaine administration increased mu-opioid receptor density in dopaminergically innervated rat brain regions, including the cingulate cortex, the nucleus accumbens, and the basolateral amygdala. The present study investigated the effects of a single day of binge-pattern cocaine administration (3 x 15 mg/kg, intraperitoneally [i.p.] at hourly intervals) on mu-opioid receptor mRNA levels in selected brain regions. Rats were sacrificed 30 min after the third injection and mRNA levels were measured by a quantitative solution hybridization RNase protection assay. Acute binge cocaine administration significantly increased mu-opioid receptor mRNA levels in the frontal cortex, nucleus accumbens, and amygdala, but not in the caudate-putamen, thalamus, hippocampus, and hypothalamus. As has been suggested for other G-protein coupled receptors, the rapid increase of MOR mRNA reported in this study might represent an adaptive response to compensate for a decrease in number of receptors following cocaine-induced opioid peptide release. PMID:10210176

  1. Interactions between chemokine and mu-opioid receptors: Anatomical findings and electrophysiological studies in the rat periaqueductal grey

    PubMed Central

    Heinisch, Silke; Palma, Jonathan; Kirby, Lynn G.

    2010-01-01

    Opioids have immunomodulatory functions and may alter susceptibility to immune disorders. Behavioral studies also indicate that chemokines, molecules expressed by immune cells, block opioid induced analgesia in the periaqueductal grey (PAG). Bi-directional heterologous desensitization of opioid and chemokine receptors has been described in cell systems. We report the anatomical and functional interactions of chemokine receptors with the mu-opioid receptor (MOR) in the rat brain. The chemokine receptors, CXCR4 and CX3CR1, as well as their chemokine substrates, CXCL12 and CX3CL1, are widely expressed in the central nervous system (CNS). Immunohistochemical techniques were utilized to investigate MOR-CXCR4 and MOR-CX3CR1 receptor colocalization in multiple brain areas. Our results demonstrate co-expression of these receptors on individual neurons in several regions including cingulate cortex, hippocampus and PAG, suggesting functional receptor interactions. Whole-cell patch-clamp recordings of PAG neurons in a rat brain slice preparation were used to examine morphine or chemokine (CXCL12, CX3CL1) effects alone or in combination on neuronal membrane properties. Morphine (10 µM) hyperpolarized and reduced input resistance of PAG neurons. CXCL12 and CX3CL1 (10 nM) had no impact on either parameter. In the presence of CXCL12, morphine’s electrophysiological effects were blocked in all neurons, whereas with CX3CL1, morphine’s effects were blocked in 57% of neurons. The data provide electrophysiological evidence for MOR-CXCR4 and MOR-CX3CR1 heterologous desensitization in the PAG at the single cell level. These interactions may contribute to the limited utility of opioid analgesics for inflammatory pain treatment and supports chemokines as neuromodulators. PMID:20974247

  2. Exposure to ethanol on prenatal days 19-20 increases ethanol intake and palatability in the infant rat: involvement of kappa and mu opioid receptors.

    PubMed

    Díaz-Cenzano, Elena; Gaztañaga, Mirari; Gabriela Chotro, M

    2014-09-01

    Prenatal exposure to ethanol on gestation Days 19-20, but not 17-18, increases ethanol acceptance in infant rats. This effect seems to be a conditioned response acquired prenatally, mediated by the opioid system, which could be stimulated by ethanol's pharmacological properties (mu-opioid receptors) or by a component of the amniotic fluid from gestation-day 20 (kappa-inducing factor). The latter option was evaluated administering non-ethanol chemosensory stimuli on gestation Days 19-20 and testing postnatal intake and palatability. However, prenatal exposure to anise or vanilla increased neither intake nor palatability of these tastants on postnatal Day 14. In experiment 2, the role of ethanol's pharmacological effect was tested by administering ethanol and selective antagonists of mu and kappa opioid receptors prenatally. Blocking the mu-opioid receptor system completely reversed the effects on intake and palatability, while antagonizing kappa receptors only partially reduced the effects on palatability. This suggests that the pharmacological effect of ethanol on the fetal mu opioid system is the appetitive reinforcer, which induces the prenatally conditioned preference detected in the preweanling period. PMID:24037591

  3. Targeted Expression of Mu-Opioid Receptors in a Subset of Striatal Direct-Pathway Neurons Restores Opiate Reward

    PubMed Central

    Cui, Yijun; Ostlund, Sean B.; James, Alex; Park, Chang Sin; Ge, Weihong; Roberts, Kristofer W.; Mittal, Nitish; Murphy, Niall P.; Cepeda, Carlos; Kieffer, Brigitte L.; Levine, Michael S.; Jentsch, J. David; Walwyn, Wendy M.; Sun, Yi E.; Evans, Christopher J.; Maidment, Nigel T.; Yang, X. William

    2014-01-01

    SUMMARY Mu-Opioid Receptors (MOR) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a novel conditional BAC rescue strategy to show that mice with targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restore opiate reward, opiate-induced striatal dopamine release, and partially restore motivation to self-administer opiates. However, they lack opiate analgesia or withdrawal. Importantly, we used Cre-mediated deletion of the rescued MOR transgene to establish that striatal, rather than a few extrastriatal sites of MOR transgene expression, is needed for the restoration of opiate reward. Together, our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a novel intersectional genetic approach to dissect neurocircuit-specific gene function in vivo. PMID:24413699

  4. Mu-Opioid Receptor Polymorphisms and Breast Cancer in a Korean Female Adult Population: A Retrospective Study

    PubMed Central

    Oh, Chung-Sik; Lee, Seung-Hyun; Yoo, Young-Bum; Yang, Jung-Hyun

    2016-01-01

    Distribution of A118G single nucleotide polymorphism (SNP) in the mu-opioid receptor 1 gene (OPRM1) differs with ethnicity. We assessed the distribution of this SNP in Korean women with breast cancer and compared it with that in women of other ethnicities with breast cancer. Distribution of SNP genotypes was as follows: 49.8% for AG genotype, 40.6% for AA genotype, and 9.6% for GG genotype. Logistic regression analysis showed a negative association between the presence of the G allele at position 118 of OPRM1 and breast cancer in the studied population (odds ratios [OR], 0.635; p=0.002). However, the AG and GG genotypes were not associated with breast cancer in the studied population (OR, 0.719; p=0.130). The proportions of the AG and GG genotypes of the OPRM1 SNP were higher in Korean women with breast cancer than in those of other ethnicities. PMID:27382398

  5. N-methyl-D-aspartate receptors and large conductance calcium-sensitive potassium channels inhibit the release of opioid peptides that induce mu-opioid receptor internalization in the rat spinal cord.

    PubMed

    Song, B; Marvizón, J C G

    2005-01-01

    Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn

  6. Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects.

    PubMed

    Majumdar, Susruta; Grinnell, Steven; Le Rouzic, Valerie; Burgman, Maxim; Polikar, Lisa; Ansonoff, Michael; Pintar, John; Pan, Ying-Xian; Pasternak, Gavril W

    2011-12-01

    Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile. PMID:22106286

  7. Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects

    PubMed Central

    Majumdar, Susruta; Grinnell, Steven; Le Rouzic, Valerie; Burgman, Maxim; Polikar, Lisa; Ansonoff, Michael; Pintar, John; Pan, Ying-Xian; Pasternak, Gavril W.

    2011-01-01

    Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile. PMID:22106286

  8. Direct association of Mu-opioid and NMDA glutamate receptors supports their cross-regulation: molecular implications for opioid tolerance.

    PubMed

    Garzón, Javier; Rodríguez-Muñoz, María; Sánchez-Blázquez, Pilar

    2012-09-01

    In the nervous system, the interaction of opioids like morphine and its derivatives, with the G protein-coupled Mu-opioid receptor (MOR) provokes the development of analgesic tolerance, as well as physical dependence. Tolerance implies that increasing doses of the drug are required to achieve the same effect, a phenomenon that contributes significantly to the social problems surrounding recreational opioid abuse. In recent years, our understanding of the mechanisms that control MOR function in the nervous system, and that eventually produce opioid tolerance, has increased greatly. Pharmacological studies have identified a number of signaling proteins involved in morphine-induced tolerance, including the N-methyl-D-aspartate acid glutamate receptor (NMDAR), nitric oxide synthase (NOS), protein kinase C (PKC), protein kinase A (PKA), calcium (Ca²⁺)/calmodulin (CaM)-dependent kinase II (CaMKII), delta-opioid receptor (DOR) and the regulators of G-protein signaling (RGS) proteins. There is general agreement on the critical role of the NMDAR/nNOS/CaMKII pathway in this process, which is supported by the recent demonstration of a physical association between MORs and NMDARs in post-synaptic structures. Indeed, it is feasible that treatments that diminish morphine tolerance may target distinct elements within the same regulatory MOR-NMDAR pathway. Accordingly, we propose a model that incorporates the most relevant signaling components implicated in opioid tolerance in which, certain signals originating from the activated MOR are perceived by the associated NMDAR, which in turn exerts a negative feedback effect on MOR signaling. MOR- and NMDAR-mediated signals work together in a sequential and interconnected manner to ultimately induce MOR desensitization. Future studies of these phenomena should focus on adding further components to this signaling pathway in order to better define the mechanism underlying MOR desensitization in neural cells. PMID:22920535

  9. Association of Smoking with Mu- Opioid Receptor Availability Before and During Naltrexone Blockade in Alcohol-Dependent Subjects

    PubMed Central

    Weerts, Elise M.; Wand, Gary S.; Kuwabara, Hiroto; Xu, Xiaoqiang; Frost, J.James; Wong, Dean F.; McCaul, Mary E.

    2012-01-01

    Persons with a history of alcohol dependence are more likely to use tobacco and to meet criteria for nicotine dependence compared to social drinkers or nondrinkers. The high levels of comorbidity of nicotine and alcohol use and dependence are thought to be related to interactions between nicotinic, opioid and dopamine receptors in mesolimbic regions. The current study examined whether individual differences in regional mu-opioid receptor (MOR) availability were associated with tobacco use, nicotine dependence, and level of nicotine craving in 25 alcohol dependent (AD) subjects. AD subjects completed an inpatient protocol, which included medically supervised alcohol withdrawal, monitored alcohol abstinence, transdermal nicotine maintenance (21 mg/day), and Positron Emission Tomography (PET) imaging using the MOR agonist [11C]-carfentanil (CFN) before (basal scan) and during treatment with 50 mg/day naltrexone (naltrexone scan). Subjects who had higher scores on the Fagerström Nicotine Dependence Test had significantly lower basal scan binding potential (BPND) across mesolimbic regions including the amygdala, cingulate, globus pallidus, thalamus and insula. Likewise, the number of cigarettes per day was negatively associated with basal scan BPND in mesolimbic regions Higher nicotine craving was significantly associated with lower BPND in amygdala, globus pallidus, putamen, thalamus and ventral striatum. Although blunted during naltrexone treatment, the negative association was maintained for nicotine dependence and cigarettes per day, but not for nicotine craving. These findings suggest that intensity of cigarette smoking and severity of nicotine dependence symptoms are systematically related to reduced BPND across multiple brain regions in AD subjects. PMID:23252742

  10. Dalargin and [Cys-(O2NH2)]2 analogues of enkephalins and their selectivity for mu opioid receptors.

    PubMed

    Pencheva, N; Ivancheva, C; Dimitrov, E; Bocheva, A; Radomirov, R

    1995-07-01

    1. Effects of the enkephalins Met-enk (M) and Leu-enk (L), of two newly synthesized analogues--[Cys-(O2NH2)]2-Met-enk (CM) and [Cys-(O2NH2)]2-Leu-enk (CL)--and of a hexapeptide--D-Ala2-Leu5-Arg6 (Dalargin; DL) on the spontaneous and electrically stimulated activity were examined with respect to their selectivity for the mu opioid receptors in the longitudinal layer of guinea pig ileum. 2. M and CM exerted relaxing and contractile effects on the spontaneous contractile activity while L, CL and DL produced only relaxation. The order of potency towards the relaxatory phase was DL > M > CM > L > CL and towards the contractile phase CM > M. 3. The effects of enkephalins on the spontaneous activity were naloxone and TTX sensitive except for the contractile phase of M and CM which persisted in the presence of TTX. NO was not involved in the neurotransmission of the relaxatory responses, while the blockade of alpha and beta adrenoceptors showed the participation of adrenergic mechanisms. Relaxation and contraction induced by enkephalins could not be directly attributed to cholinergic neurotransmission. 4. The naloxone-sensitive and concentration-dependent inhibitory effects of enkephalins and their analogues on the electrically stimulated cholinergic contractions were established. The order of the relative potency of opioids was: DL-3.8; M-1.0; L-0.4; CM-0.01; CL-0.005. 5. These data indicated that the D-Ala2 substitution and lengthening of the peptide chain by Arg6 in the molecule of L increased the potency at the mu opiate receptors, while the substitution in position 2 with Cys-(O2NH2) in the molecule of M and L yielded a less potent and selective mu agonists. PMID:7635255

  11. It’s MORe exciting than mu: crosstalk between mu opioid receptors and glutamatergic transmission in the mesolimbic dopamine system

    PubMed Central

    Chartoff, Elena H.; Connery, Hilary S.

    2014-01-01

    Opioids selective for the G protein-coupled mu opioid receptor (MOR) produce potent analgesia and euphoria. Heroin, a synthetic opioid, is considered one of the most addictive substances, and the recent exponential rise in opioid addiction and overdose deaths has made treatment development a national public health priority. Existing medications (methadone, buprenorphine, and naltrexone), when combined with psychosocial therapies, have proven efficacy in reducing aspects of opioid addiction. Unfortunately, these medications have critical limitations including those associated with opioid agonist therapies (e.g., sustained physiological dependence and opioid withdrawal leading to high relapse rates upon discontinuation), non-adherence to daily dosing, and non-renewal of monthly injection with extended-release naltrexone. Furthermore, current medications fail to ameliorate key aspects of addiction such as powerful conditioned associations that trigger relapse (e.g., cues, stress, the drug itself). Thus, there is a need for developing novel treatments that target neural processes corrupted with chronic opioid use. This requires a basic understanding of molecular and cellular mechanisms underlying effects of opioids on synaptic transmission and plasticity within reward-related neural circuits. The focus of this review is to discuss how crosstalk between MOR-associated G protein signaling and glutamatergic neurotransmission leads to immediate and long-term effects on emotional states (e.g., euphoria, depression) and motivated behavior (e.g., drug-seeking, relapse). Our goal is to integrate findings on how opioids modulate synaptic release of glutamate and postsynaptic transmission via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors in the nucleus accumbens and ventral tegmental area with the clinical (neurobehavioral) progression of opioid dependence, as well as to identify gaps in knowledge that can be addressed in future studies

  12. Mu opioid receptor up-regulation and participation in excitability of hippocampal pyramidal cell electrophysiology

    SciTech Connect

    Moudy, A.M.

    1988-01-01

    Chronic administration of opiate antagonists to rats results in up-regulation of their brain opioid receptors. Using subcellular fractionation techniques, brain opioid receptors were resolved into two membrane populations, one associated with synaptic plasma membranes (SPM) and the other enriched in smooth endoplasmic reticulum and Golgi (microsomes). This study addressed in part the question of whether an antagonist induces up-regulation uniformly in these two populations. Rats were administered naltrexone by subcutaneously implanted osmotic minipumps. Forebrain mu receptor levels were determined by homologous displacement of ({sup 3}H)D-ala{sup 2}-mePhe{sup 4}-gly-ol{sup 5}-enkephalin (DAGO) followed by computer estimation of binding parameters. Receptor levels in crude membranes rose 77% after treatment. Microsomes displayed a 92% increase, a two-fold greater change than in SPMs (51%). These results establish that naltrexone induces up-regulation of both membrane populations; and that microsomal and SPM receptors represent discrete populations of intracellular and cell surface sites, respectively. Binding experiments on isolated hippocampi also demonstrated up-regulation (71%) of mu receptors. To demonstrate up-regulation of opioid receptors electrophysiologically, hippocampal slices were prepared from rats which had been chronically treated with naltrexone. After superfusion with DAGO, these slices showed a 42% greater population spike output than controls in response to the same EPSP input. Hippocampi from animals treated for two weeks showed an additional increase in sensitivity. The results support a disinhibitory role for opioids in pyramidal cell hyper-excitability. More importantly, they demonstrate a significant physiological correlate to opioid receptor up-regulation.

  13. The Mu Opioid Receptor A118G Gene Polymorphism Moderates Effects of Trait Anger-Out on Acute Pain Sensitivity

    PubMed Central

    Bruehl, Stephen; Chung, Ok Y.; Burns, John W.

    2008-01-01

    Both trait anger-in (managing anger through suppression) and anger-out (managing anger through direct expression) are related to pain responsiveness, but only anger-out effects involve opioid mechanisms. Preliminary work suggested the effects of anger-out on post-operative analgesic requirements were moderated by the A118G single nucleotide polymorphism of the mu opioid receptor gene. This study further explored these potential genotype X phenotype interactions as they impact acute pain sensitivity. Genetic samples and measures of anger-in and anger-out were obtained in 87 subjects (from three studies) who participated in controlled laboratory acute pain tasks (ischemic, finger pressure, thermal). McGill Pain Questionnaire (MPQ) Sensory and Affective ratings for each pain task were standardized within studies, aggregated across pain tasks, and combined for analyses. Significant anger-out X A118G interactions were observed (p’s<.05). Simple effects tests for both pain measures revealed that whereas anger-out was nonsignificantly hyperalgesic in subjects homozygous for the wild-type allele, anger-out was significantly hypoalgesic in those with the variant G allele (p’s<.05). For the MPQ-Affective measure, this interaction arose both from low pain sensitivity in high anger-out subjects with the G allele and heightened pain sensitivity in low anger-out subjects with the G allele relative to responses in homozygous wild-type subjects. No genetic moderation was observed for anger-in, although significant main effects on MPQ-Affective ratings were noted (p<.005). Anger-in main effects were due to overlap with negative affect, but anger-out X A118G interactions were not, suggesting unique effects of expressive anger regulation. Results support opioid-related genotype X phenotype interactions involving trait anger-out. PMID:18579306

  14. Effects of defeat stress on behavioral flexibility in males and females: modulation by the mu-opioid receptor

    PubMed Central

    Laredo, Sarah A.; Steinman, Michael Q.; Robles, Cindee F.; Ferrer, Emilio; Ragen, Benjamin J.; Trainor, Brian C.

    2014-01-01

    Behavioral flexibility is a component of executive functioning that allows individuals to adapt to changing environmental conditions. Independent lines of research indicate that the mu opioid receptor (MOR) is an important mediator of behavioral flexibility and responses to psychosocial stress. The current study bridges these two lines of research and tests the extent to which social defeat and MOR affect behavioral flexibility and whether sex moderates these effects in California mice (Peromyscus californicus). Males and females assigned to social defeat or control conditions were tested in a Barnes maze. In males, defeat impaired behavioral flexibility but not acquisition. Female performance was unaffected by defeat. MOR binding in defeated and control mice in the orbitofrontal cortex (OFC), striatum, and hippocampus was examined via autoradiography. Stressed males had reduced MOR binding in the OFC whereas females were unaffected. The MOR antagonist beta-funaltrexamine (1 mg/kg) impaired performance in males naïve to defeat during the reversal phase but had no effect on females. Finally, we examined the effects of the MOR agonist morphine (2.5, 5 mg/kg) on stressed mice. As expected, morphine improved behavioral flexibility in stressed males. The stress-induced deficits in behavioral flexibility in males are consistent with a proactive coping strategy, including previous observations that stressed male California mice exhibit strong social approach and aggression. Our pharmacological data suggest that a down-regulation of MOR signaling in males may contribute to sex differences in behavioral flexibility following stress. This is discussed in the framework of coping strategies for individuals with mood disorders. PMID:25615538

  15. Effects of defeat stress on behavioral flexibility in males and females: modulation by the mu-opioid receptor.

    PubMed

    Laredo, Sarah A; Steinman, Michael Q; Robles, Cindee F; Ferrer, Emilio; Ragen, Benjamin J; Trainor, Brian C

    2015-02-01

    Behavioral flexibility is a component of executive functioning that allows individuals to adapt to changing environmental conditions. Independent lines of research indicate that the mu opioid receptor (MOR) is an important mediator of behavioral flexibility and responses to psychosocial stress. The current study bridges these two lines of research and tests the extent to which social defeat and MOR affect behavioral flexibility and whether sex moderates these effects in California mice (Peromyscus californicus). Males and females assigned to social defeat or control conditions were tested in a Barnes maze. In males, defeat impaired behavioral flexibility but not acquisition. Female performance was unaffected by defeat. MOR binding in defeated and control mice in the orbitofrontal cortex (OFC), striatum and hippocampus was examined via autoradiography. Stressed males had reduced MOR binding in the OFC whereas females were unaffected. The MOR antagonist beta-funaltrexamine (1 mg/kg) impaired performance in males naïve to defeat during the reversal phase but had no effect on females. Finally, we examined the effects of the MOR agonist morphine (2.5 and 5 mg/kg) on stressed mice. As expected, morphine improved behavioral flexibility in stressed males. The stress-induced deficits in behavioral flexibility in males are consistent with a proactive coping strategy, including previous observations that stressed male California mice exhibit strong social approach and aggression. Our pharmacological data suggest that a down-regulation of MOR signaling in males may contribute to sex differences in behavioral flexibility following stress. This is discussed in the framework of coping strategies for individuals with mood disorders. PMID:25615538

  16. Mediation of buprenorphine analgesia by a combination of traditional and truncated mu opioid receptor splice variants.

    PubMed

    Grinnell, Steven G; Ansonoff, Michael; Marrone, Gina F; Lu, Zhigang; Narayan, Ankita; Xu, Jin; Rossi, Grace; Majumdar, Susruta; Pan, Ying-Xian; Bassoni, Daniel L; Pintar, John; Pasternak, Gavril W

    2016-10-01

    Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit β-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors. PMID:27223691

  17. Analgesic tolerance of opioid agonists in mutant mu-opioid receptors expressed in sensory neurons following intrathecal plasmid gene delivery

    PubMed Central

    2013-01-01

    Background Phosphorylation sites in the C-terminus of mu-opioid receptors (MORs) are known to play critical roles in the receptor functions. Our understanding of their participation in opioid analgesia is mostly based on studies of opioid effects on mutant receptors expressed in in vitro preparations, including cell lines, isolated neurons and brain slices. The behavioral consequences of the mutation have not been fully explored due to the complexity in studies of mutant receptors in vivo. To facilitate the determination of the contribution of phosphorylation sites in MOR to opioid-induced analgesic behaviors, we expressed mutant and wild-type human MORs (hMORs) in sensory dorsal root ganglion (DRG) neurons, a major site for nociceptive (pain) signaling and determined morphine- and the full MOR agonist, DAMGO,-induced effects on heat-induced hyperalgesic behaviors and potassium current (IK) desensitization in these rats. Findings A mutant hMOR DNA with the putative phosphorylation threonine site at position 394 replaced by an alanine (T394A), i.e., hMOR-T, or a plasmid containing wild type hMOR (as a positive control) was intrathecally delivered. The plasmid containing GFP or saline was used as the negative control. To limit the expression of exogenous DNA to neurons of DRGs, a neuron-specific promoter was included in the plasmid. Following a plasmid injection, hMOR-T or hMOR receptors were expressed in small and medium DRG neurons. Compared with saline or GFP rats, the analgesic potency of morphine was increased to a similar extent in hMOR-T and hMOR rats. Morphine induced minimum IK desensitization in both rat groups. In contrast, DAMGO increased analgesic potency and elicited IK desensitization to a significantly less extent in hMOR-T than in hMOR rats. The development and extent of acute and chronic tolerance induced by repeated morphine or DAMGO applications were not altered by the T394A mutation. Conclusions These results indicate that phosphorylation of T394

  18. Cell-Autonomous Regulation of Mu-Opioid Receptor Recycling by Substance P

    PubMed Central

    Bowman, Shanna L.; Soohoo, Amanda L.; Shiwarski, Daniel J.; Schulz, Stefan; Pradhan, Amynah A.; Puthenveedu, Manojkumar A.

    2015-01-01

    SUMMARY How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropep-tide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the muopioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeo-static interaction between the pain and analgesic systems. PMID:25801029

  19. Role of the mu opioid receptor in opioid modulation of immune function

    PubMed Central

    Ninković, Jana; Roy, Sabita

    2014-01-01

    SUMMARY Endogenous opioids are synthesized in vivo in order to modulate pain mechanisms and inflammatory pathways. Endogenous and exogenous opioids mediate analgesia in response to painful stimuli by binding to opioid receptors on neuronal cells. However, wide distribution of opioid receptors on tissues and organ systems outside the CNS, such as the cells of the immune system, indicate that opioids are capable of exerting additional effects in the periphery, such as immunomodulation. The increased prevalence of infections in opioid abusers based epidemiological studies further highlights the immunosuppressive effects of opioids. In spite of their many debilitating side effects, prescription opioids remain a gold standard for treatment of chronic pain. Therefore, given the prevalence of opioid use and abuse, opioid mediated immune suppression presents a serious concern in our society today. It is imperative to understand the mechanisms by which exogenous opioids modulate immune processes. In this review we will discuss the role of opioid receptors and their ligands in mediating immune suppressive functions. We will summarize recent studies on direct and indirect opioid modulation of the cells of the immune system as well as the role of opioids in exacerbation of certain disease states. PMID:22170499

  20. Neonatal Administration of Thimerosal Causes Persistent Changes in Mu Opioid Receptors in the Rat Brain

    PubMed Central

    Olczak, Mieszko; Duszczyk, Michalina; Mierzejewski, Pawel; Bobrowicz, Teresa

    2010-01-01

    Thimerosal added to some pediatric vaccines is suspected in pathogenesis of several neurodevelopmental disorders. Our previous study showed that thimerosal administered to suckling rats causes persistent, endogenous opioid-mediated hypoalgesia. Here we examined, using immunohistochemical staining technique, the density of μ-opioid receptors (MORs) in the brains of rats, which in the second postnatal week received four i.m. injections of thimerosal at doses 12, 240, 1,440 or 3,000 μg Hg/kg. The periaqueductal gray, caudate putamen and hippocampus were examined. Thimerosal administration caused dose-dependent statistically significant increase in MOR densities in the periaqueductal gray and caudate putamen, but decrease in the dentate gyrus, where it was accompanied by the presence of degenerating neurons and loss of synaptic vesicle marker (synaptophysin). These data document that exposure to thimerosal during early postnatal life produces lasting alterations in the densities of brain opioid receptors along with other neuropathological changes, which may disturb brain development. PMID:20803069

  1. Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells

    SciTech Connect

    Mouledous, Lionel

    2008-08-15

    *: Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF{sub 2}) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF{sub 2} receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

  2. Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed Efficacy Mu Opioid Receptor (MOR) Agonist/Delta Opioid Receptor (DOR) Antagonist Ligands

    PubMed Central

    Mosberg, Henry I.; Yeomans, Larisa; Harland, Aubrie A.; Bender, Aaron M.; Sobczyk-Kojiro, Katarzyna; Anand, Jessica P.; Clark, Mary J.; Jutkiewicz, Emily M.; Traynor, John R.

    2013-01-01

    We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a mu opioid receptor (MOR) agonist, delta opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance. PMID:23419026

  3. [Interactions of peripheral mu-opioid receptors and K(ATP)-channels in regulation of cardiac electrical stability in ischemia, reperfusion, and postinfarction cardiosclerosis].

    PubMed

    Maslov, L N; Krylatov, A V; Naryzhaia, N V; Solenkova, N V; Lishmanov, A Iu; Bogomaz, S A; Gross, G J; Stefano, J B; Loktiushina, B A

    2002-07-01

    It has been shown that mu-opioid receptor stimulation by intravenous administration of the selective mu receptor agonist DALDA in a dose of 0.1 mg/kg prevented ischemic and reperfusion arrhythmias in rats subjected to coronary artery occlusion (10 min) and reperfusion (10 min), and also increased the ventricular fibrillation threshold in rats with postinfarction cardiac fibrosis. These effects were abolished by pre-treatment with the selective mu receptor antagonist CTAP in a dose of 0.5 mg/kg or by prior injection of the opioid receptor antagonist naloxone methiodide (2 mg/kg) which does not penetrate the blood-braib barrier. Both antagonists by themselves had no effect on the incidence of occlusion or reperfusion-induced arrhythmias or on the ventricular fibrillation threshold. Pre-treatment with ATP-sensitive K+ channel (KATP channel) blocker glibenclamide in a dose of 0.3 mg/kg completely abolished the antiarrhythmic effect of DALDA. We believe that DALDA prevents occurrence of electrical instability during ischemia and reperfusion and increases the ventricular fibrillation threshold in rats with postinfarction cardiac fibrosis via stimulation of peripheral mu-opioid receptor which appear to be coupled to the KATP channel. PMID:12238351

  4. Morphine-induced trafficking of a mu-opioid receptor interacting protein in rat locus coeruleus neurons.

    PubMed

    Jaremko, Kellie M; Thompson, Nicholas L; Reyes, Beverly A S; Jin, Jay; Ebersole, Brittany; Jenney, Christopher B; Grigson, Patricia S; Levenson, Robert; Berrettini, Wade H; Van Bockstaele, Elisabeth J

    2014-04-01

    Opiate addiction is a devastating health problem, with approximately 2million people currently addicted to heroin or non-medical prescription opiates in the United States alone. In neurons, adaptations in cell signaling cascades develop following opioid actions at the mu opioid receptor (MOR). A novel putative target for intervention involves interacting proteins that may regulate trafficking of MOR. Morphine has been shown to induce a re-distribution of a MOR-interacting protein Wntless (WLS, a transport molecule necessary for secretion of neurotrophic Wnt proteins), from cytoplasmic to membrane compartments in rat striatal neurons. Given its opiate-sensitivity and its well-characterized molecular and cellular adaptations to morphine exposure, we investigated the anatomical distribution of WLS and MOR in the rat locus coeruleus (LC)-norepinephrine (NE) system. Dual immunofluorescence microscopy was used to test the hypothesis that WLS is localized to noradrenergic neurons of the LC and that WLS and MOR co-exist in common LC somatodendritic processes, providing an anatomical substrate for their putative interactions. We also hypothesized that morphine would influence WLS distribution in the LC. Rats received saline, morphine or the opiate agonist [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), and tissue sections through the LC were processed for immunogold-silver detection of WLS and MOR. Statistical analysis showed a significant re-distribution of WLS to the plasma membrane following morphine treatment in addition to an increase in the proximity of gold-silver labels for MOR and WLS. Following DAMGO treatment, MOR and WLS were predominantly localized within the cytoplasmic compartment when compared to morphine and control. In a separate cohort of rats, brains were obtained from saline-treated or heroin self-administering male rats for pulldown co-immunoprecipitation studies. Results showed an increased association of WLS and MOR following heroin exposure. As the

  5. Morphine-induced trafficking of a mu-opioid receptor interacting protein in rat locus coeruleus neurons

    PubMed Central

    Jaremko, Kellie M.; Thompson, Nicholas L.; Reyes, Beverly A. S.; Jin, Jay; Ebersole, Brittany; Jenney, Christopher B.; Grigson, Patricia S.; Levenson, Robert; Berrettini, Wade H.; Van Bockstaele, Elisabeth J.

    2014-01-01

    Opiate addiction is a devastating health problem, with approximately 2 million people currently addicted to heroin or non-medical prescription opiates in the United States alone. In neurons, adaptations in cell signaling cascades develop following opioid actions at the mu opioid receptor (MOR). A novel putative target for intervention involves interacting proteins that may regulate trafficking of MOR. Morphine has been shown to induce a re-distribution of a MOR-interacting protein Wntless (WLS, a transport molecule necessary for secretion of neurotrophic Wnt proteins), from cytoplasmic to membrane compartments in rat striatal neurons. Given its opiate-sensitivity and its well-characterized molecular and cellular adaptations to morphine exposure, we investigated the anatomical distribution of WLS and MOR in the rat locus coeruleus (LC)-norepinephrine (NE) system. Dual immunofluorescence microscopy was used to test the hypothesis that WLS is localized to noradrenergic neurons of the LC and that WLS and MOR co-exist in common LC somatodendritic processes, providing an anatomical substrate for their putative interactions. We also hypothesized that morphine would influence WLS distribution in the LC. Rats received saline, morphine or the opiate agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), and tissue sections through the LC were processed for immunogold-silver detection of WLS and MOR. Statistical analysis showed a significant re-distribution of WLS to the plasma membrane following morphine treatment in addition to an increase in the proximity of gold-silver labels for MOR and WLS. Following DAMGO treatment, MOR and WLS were predominantly localized within the cytoplasmic compartment when compared to morphine and control. In a separate cohort of rats, brains were obtained from saline-treated or heroin self-administering male rats for pulldown co-immunoprecipitation studies. Results showed an increased association of WLS and MOR following heroin exposure. As

  6. /sup 125/I-FK 33-824: a selective probe for radioautographic labeling of mu opioid receptors in the brain

    SciTech Connect

    Moyse, E.; Pasquini, F.; Quirion, R.; Beaudet, A.

    1986-03-01

    The selectivity of the Met-enkephalin analog FK 33-824 (FK) for mu opioid receptors has been, over the years, a matter of controversy. We report here pharmacological and radioautographic data demonstrating that at nanomolar concentrations. /sup 125/I-FK interacts exclusively with mu sites. (1) Specific binding of /sup 125/I-FK to rat striatal membranes is totally inhibited by mu- and/or delta-preferring ligands according to monovalent, Michaelian kinetics, with a potency proportional to the affinity of competing drugs for mu receptors. (2) Unlabeled FK competes only at high concentration with the delta-selective ligand 3H-DPLPE and according to the same kinetics as the mu-selective agonist DAGO. (3) /sup 125/I-FK generates the same regional radioautographic labeling pattern as 3H-DAGO. We conclude that when used at nanomolar concentrations /sup 125/I-FK constitutes a selective probe for the radioautographic detection of mu opioid receptors at both light and electron microscopic levels.

  7. Mu opioid receptor in spermatozoa, eggs and larvae of gilthead sea bream (Sparus Aurata) and its involvement in stress related to aquaculture.

    PubMed

    Albrizio, Maria; Guaricci, Antonio C; Milano, Serena; Macrì, Francesco; Aiudi, Giulio

    2014-08-01

    In aquaculture, unfavourable conditions experienced during early development may have strong downstream effects on the adult phenotype and fitness. Sensitivity to stress, leading to disease, reduced growth and mortality, is higher in larvae than in adult fish. In this study, conducted on sea bream (Sparus aurata), we evidenced the presence of the mu opioid receptor in gametes and larvae at different developmental stages. Moreover, we evaluated the possibility of reducing the effects of artificially produced stress, altering temperature, salinity and pH, by naloxone (an opioid antagonist) and calcium. Results evidenced that mu opioid receptor is present in larvae and in gametes of both sexes and that, during larval growth, its expression level changes accordingly; furthermore, naloxone/calcium association is efficacious in increasing the survival period of treated larvae compared to controls. We conclude that in sea bream rearing, the use of naloxone/calcium against stress can improve fish farming techniques by reducing larval mortality and consequently increasing productivity. PMID:24338156

  8. Argon blocks the expression of locomotor sensitization to amphetamine through antagonism at the vesicular monoamine transporter-2 and mu-opioid receptor in the nucleus accumbens.

    PubMed

    David, H N; Dhilly, M; Degoulet, M; Poisnel, G; Meckler, C; Vallée, N; Blatteau, J-É; Risso, J-J; Lemaire, M; Debruyne, D; Abraini, J H

    2015-01-01

    We investigated the effects of the noble gas argon on the expression of locomotor sensitization to amphetamine and amphetamine-induced changes in dopamine release and mu-opioid neurotransmission in the nucleus accumbens. We found (1) argon blocked the increase in carrier-mediated dopamine release induced by amphetamine in brain slices, but, in contrast, potentiated the decrease in KCl-evoked dopamine release induced by amphetamine, thereby suggesting that argon inhibited the vesicular monoamine transporter-2; (2) argon blocked the expression of locomotor and mu-opioid neurotransmission sensitization induced by repeated amphetamine administration in a short-term model of sensitization in rats; (3) argon decreased the maximal number of binding sites and increased the dissociation constant of mu-receptors in membrane preparations, thereby indicating that argon is a mu-receptor antagonist; (4) argon blocked the expression of locomotor sensitization and context-dependent locomotor activity induced by repeated administration of amphetamine in a long-term model of sensitization. Taken together, these data indicate that argon could be of potential interest for treating drug addiction and dependence. PMID:26151922

  9. Mu-opioid receptor activation prevents apoptosis following serum withdrawal in differentiated SH-SY5Y cells and cortical neurons via phosphatidylinositol 3-kinase.

    PubMed

    Iglesias, M; Segura, M F; Comella, J X; Olmos, G

    2003-03-01

    Opioid peptides and alkaloids exert their effects via G protein-coupled receptors (GPCRs). It has been shown that, in addition to trophic factors, some GPCRs are able to activate the phosphatidylinositol 3-kinase/Akt (PI 3-K/Akt) signal transduction pathway, thus leading to cell survival. The aim of this study was to test whether activation of mu-opioid receptors has protective effects on serum withdrawal-induced cell death and to study the possible implication of PI 3-K in this process. In SH-SY5Y neuroblastoma cells fully differentiated by exposure to retinoic acid for five days, the enkephalin derivative selective mu-agonist DAMGO (0.1-2 microM) and the alkaloid morphine (0.1-10 microM) promoted cell survival after serum deprivation (MTT and trypan blue exclusion assays), without inducing cell proliferation. These effects were fully reversed by naloxone, by the selective mu-antagonist beta-funaltrexamine (beta-FNA) and also by the specific PI 3-K inhibitor LY294002. The two agonists stimulated Akt phosphorylation and the effect was also abolished by beta-FNA and by LY294002. In mouse primary cortical neurons, DAMGO reduced the percentage of apoptosis after 6, 12, 24 and 48 h of serum withdrawal; as determined by Hoechst staining. This effect was blocked by beta-FNA, by pre-treatment with pertussis toxin and by LY294002. DAMGO also stimulated Akt phosphorylation via PI 3-K in this primary neuronal culture. Together, these results indicate that stimulation of the mu-opioid receptor promotes neuronal survival in a G(i/o)-linked, PI 3-K-dependent signaling cascade and suggest that Akt may be a key downstream kinase involved in this anti-apoptotic effect. PMID:12646285

  10. Binding and structure-activity-relation of benzo[f]isoquinoline- and norcodeinone-derivatives at mu-opioid receptors in the rat cerebral cortex.

    PubMed Central

    Freissmuth, M.; Beindl, W.; Kratzel, M.

    1993-01-01

    1. We have probed the ligand binding site of the mu-opioid receptor using a series of isoquinoline- and norcodeinone-derivatives; in these morphine- and codeine-analogues, the position of the piperidine-nitrogen as well as its mobility is altered relative to that found in morphine. 2. The mu-receptor in rat cortical membranes was labelled with [3H]-naloxone and competition experiments were carried out in the absence and presence of Gpp(NH)p and NaCl: conditions, which are associated with affinity shifts for agonists whilst antagonist affinity remains unaffected. Moving the piperidine-nitrogen closer to the phenolic ring or reducing its mobility by incorporation into an additional ring drastically decreases the affinity. 3. In contrast, we find that the piperidine-nitrogen in a distal position is well tolerated provided that additional structural criteria, in particular a phenolic hydroxyl-group and a 6 carbon ring corresponding to ring C in morphine, are met. This assumption was verified by the synthesis of WB4/PH (4aR, 10bS, 11R)-10, 11-epoxy-1, 2, 3, 4, 5, 6-hexahydro-9-hydroxy-3-methyl-4a,10b-butano- benzo[f]isochinolin-12-on(10). This compound is an agonist with an affinity comparable to that of morphine. 4. We therefore conclude that both the mobility of the piperidine nitrogen of the ligand and of its counterpart anionic site in the ligand binding pocket of the mu-opioid receptor (presumably aspartic acid) are important determinants for fruitful interaction. The mobility of the anionic site is restricted in one direction but is sufficient to bridge the 2A distance that exists between the position of the nitrogen in morphine and WB4/PH. PMID:8306082

  11. Mu-Opioid (MOP) receptor mediated G-protein signaling is impaired in specific brain regions in a rat model of schizophrenia.

    PubMed

    Szűcs, Edina; Büki, Alexandra; Kékesi, Gabriella; Horváth, Gyöngyi; Benyhe, Sándor

    2016-04-21

    Schizophrenia is a complex mental health disorder. Clinical reports suggest that many patients with schizophrenia are less sensitive to pain than other individuals. Animal models do not interpret schizophrenia completely, but they can model a number of symptoms of the disease, including decreased pain sensitivities and increased pain thresholds of various modalities. Opioid receptors and endogenous opioid peptides have a substantial role in analgesia. In this biochemical study we investigated changes in the signaling properties of the mu-opioid (MOP) receptor in different brain regions, which are involved in the pain transmission, i.e., thalamus, olfactory bulb, prefrontal cortex and hippocampus. Our goal was to compare the transmembrane signaling mediated by MOP receptors in control rats and in a recently developed rat model of schizophrenia. Regulatory G-protein activation via MOP receptors were measured in [(35)S]GTPγS binding assays in the presence of a highly selective MOP receptor peptide agonist, DAMGO. It was found that the MOP receptor mediated activation of G-proteins was substantially lower in membranes prepared from the 'schizophrenic' model rats than in control animals. The potency of DAMGO to activate MOP receptor was also decreased in all brain regions studied. Taken together in our rat model of schizophrenia, MOP receptor mediated G-proteins have a reduced stimulatory activity compared to membrane preparations taken from control animals. The observed distinct changes of opioid receptor functions in different areas of the brain do not explain the augmented nociceptive threshold described in these animals. PMID:26946106

  12. Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.

    PubMed

    Chen, C; Yin, J; Riel, J K; DesJarlais, R L; Raveglia, L F; Zhu, J; Liu-Chen, L Y

    1996-08-30

    We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor. PMID:8702924

  13. MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens

    PubMed Central

    Jin, Jay; Wong, Victoria; Kittanakom, Saranya; Ferraro, Thomas N.; Stagljar, Igor; Levenson, Robert

    2013-01-01

    The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction. PMID:23840749

  14. Preferential cytoplasmic localization of delta-opioid receptors in rat striatal patches: comparison with plasmalemmal mu-opioid receptors.

    PubMed

    Wang, H; Pickel, V M

    2001-05-01

    The activation of delta-opioid receptors (DORs) in the caudate-putamen nucleus (CPN) produces regionally distinct changes in motor functions, many of which are also influenced by opioids active at micro-opioid receptors (MORs). These actions most likely occur in MOR-enriched patch compartments in the CPN. To determine the functional sites for DOR activation and potential interactions involving MOR in these regions, immunoperoxidase and immunogold-silver labeling methods were applied reversibly for the ultrastructural localization of DOR and MOR in single rat brain sections containing patches of the CPN. DOR immunoreactivity was commonly seen within the cytoplasm of spiny and aspiny neurons, many of which also expressed MOR. In dendrites and spines, DOR labeling was preferentially localized to membranes of the smooth endoplasmic reticulum and spine apparatus, whereas MOR showed a prominent plasmalemmal distribution. DOR- and/or MOR-labeled spines received asymmetric, excitatory synapses, some of which showed notable perforations, suggesting the involvement of these receptors in activity-dependent synaptic plasticity. DORs were more frequently detected than were MORs within axon terminals that formed either asymmetric synapses with spine heads or symmetric synapses with spine necks. Our results suggest that in striatal patches, DORs, often in cooperation with MORs, play a direct modulatory role in controlling the postsynaptic excitability of spines, whereas presynaptic neurotransmitter release onto spines is mainly influenced by DOR activation. In comparison with MOR, the prevalent association of DOR with cytoplasmic organelles that are involved in intracellular trafficking of cell surface proteins suggests major differences in availability of these receptors to extracellular opioids. PMID:11312309

  15. Co-development of early adolescent alcohol use and depressive feelings: The role of the mu-opioid receptor A118G polymorphism.

    PubMed

    Kleinjan, Marloes; Rozing, Mayke; Engels, Rutger C M E; Verhagen, Maaike

    2015-08-01

    Alcohol use and depressive feelings are often related among early adolescents. However, the nature and underlying mechanisms of this association are not yet clear. The aim of this study was to investigate the co-development of alcohol use and depressive feelings over time and to examine the effects of the mu-opioid receptor (OPRM1) A118G genotype on such co-development. Data from a five-wave longitudinal, genetically informed survey study, with intervals of 4 months among a group of 739 normative early adolescents (12-13 years of age at baseline), were analyzed using a dual latent growth curve approach. OPRM1 status was evaluated from saliva-derived DNA samples. The results indicated a positive association between alcohol use and depressive feelings both at the initial levels and over time, indicating co-development in early adolescence. Compared to OPRM1 118G carriers, homozygous 118A carriers showed a greater increase in frequency of alcohol use and higher levels of depressive feelings over time. Evidence for co-development was only found within the group of homozygous 118A carriers, whereas in OPRM1 118G carriers the development of alcohol use and depressive feelings over time were not significantly associated. These results highlight the potential of OPRM1 as a common etiological factor for the development of alcohol use and depressive feelings in early adolescence. PMID:25215437

  16. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve

    PubMed Central

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  17. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve.

    PubMed

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  18. Methamphetamine-induced stereotypy correlates negatively with patch-enhanced prodynorphin and arc mRNA expression in the rat caudate putamen: the role of mu opioid receptor activation.

    PubMed

    Horner, Kristen A; Noble, Erika S; Gilbert, Yamiece E

    2010-06-01

    Amphetamines induce stereotypy, which correlates with patch-enhanced c-Fos expression the patch compartment of caudate putamen (CPu). Methamphetamine (METH) treatment also induces patch-enhanced expression of prodynorphin (PD), arc and zif/268 in the CPu. Whether patch-enhanced activation of any of these genes correlates with METH-induced stereotypy is unknown, and the factors that contribute to this pattern of expression are poorly understood. Activation of mu opioid receptors, which are expressed by the neurons of the patch compartment, may underlie METH-induced patch-enhanced gene expression and stereotypy. The current study examined whether striatal mu opioid receptor blockade altered METH-induced stereotypy and patch-enhanced gene expression, and if there was a correlation between the two responses. Animals were intrastriatally infused with the mu antagonist CTAP (10 microg/microl), treated with METH (7.5 mg/kg, s.c.), placed in activity chambers for 3h, and then sacrificed. CTAP pretreatment attenuated METH-induced increases in PD, arc and zif/268 mRNA expression and significantly reduced METH-induced stereotypy. Patch-enhanced PD and arc mRNA expression in the dorsolateral CPu correlated negatively with METH-induced stereotypy. These data indicate that mu opioid receptor activation contributes to METH-induced gene expression in the CPu and stereotypy, and that patch-enhanced PD and arc expression may be a homeostatic response to METH treatment. PMID:20298714

  19. The functional expression of mu opioid receptors on sensory neurons is developmentally regulated; morphine analgesia is less selective in the neonate.

    PubMed

    Nandi, Reema; Beacham, Daniel; Middleton, Jacqueta; Koltzenburg, Martin; Howard, Richard F; Fitzgerald, Maria

    2004-09-01

    Opioid requirements in neonatal patients are reported to be lower than older infants and this may be a reflection of the developmental regulation of opioid receptors. In this study we have investigated the postnatal regulation of Mu opioid receptor (MOR) function in both rat lumbar dorsal root ganglion (DRG) cultures and behavioural mechanical and thermal reflex tests in rat pups. Immunostaining with MOR and selective neurofilament (NF200) antibodies was combined with calcium imaging of MOR function in cultured neonatal and adult rat dorsal root ganglion cells. Calcium imaging showed that a significantly greater number of neonatal DRG neurons expressed functional MOR compared to adult (56.5+/-3.4 versus 39.9+/-1.5%, n=8, mean+/-SEM, P<0.001). This expression is confined to the large, neurofilament positive sensory neurons, while expression in small, nociceptive, neurofilament negative neurons remains unchanged. Sensory threshold testing in rat pups showed that the analgesic potency of systemic morphine to mechanical stimulation is significantly greater in the neonate and declines with postnatal age. Morphine analgesic potency in thermal nociceptive tests did not change with postnatal age. These experiments show that the MOR expressed on large DRG neurons in neonates are functional and are subject to postnatal developmental regulation. This changing functional receptor profile is consistent with greater morphine potency in mechanical, but not thermal, sensory tests in young animals. These results have important clinical implications for the use of morphine in neonates and provide a possible explanation for the differences in morphine requirements observed in the youngest patients. PMID:15327807

  20. Design, Synthesis, and Biological Evaluation of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4’-pyridyl)carboxamido]morphinan Derivatives as Peripheral Selective Mu Opioid Receptor Agents

    PubMed Central

    Yuan, Yunyun; Elbegdorj, Orgil; Chen, Jianyang; Akubathini, Shashidhar K.; Zhang, Feng; Stevens, David L.; Beletskaya, Irina O.; Scoggins, Krista L.; Zhang, Zhenxian; Gerk, Phillip M.; Selley, Dana E.; Akbarali, Hamid I.; Dewey, William L.; Zhang, Yan

    2012-01-01

    Peripheral selective mu opioid receptor (MOR) antagonists could alleviate the symptoms of opioid-induced constipation (OIC) without compromising the analgesic effect of opioids. However, a variety of adverse effects were associated with them, partially due to their relatively low MOR selectivity. NAP, a 6β-N-4'-pyridyl substituted naltrexamine derivative, was identified previously as a potent and highly selective MOR antagonist mainly acting within the peripheral nervous system. The noticeable diarrhea associated with it prompted the design and synthesis of its analogues in order to study its structure activity relationship. Among them, compound 8 showed improved pharmacological profiles compared to the original lead, acting mainly at peripheral while increasing the intestinal motility in morphine-pelleted mice (ED50=0.03 mg/kg). The slight decrease of the ED50 compared to the original lead was well compensated by the unobserved adverse effect. Hence, this compound seems to be a more promising lead to develop novel therapeutic agents toward OIC. PMID:23116124

  1. A role for kappa-, but not mu-opioid, receptor activation in acute food deprivation-induced reinstatement of heroin seeking in rats.

    PubMed

    Sedki, Firas; Eigenmann, Karine; Gelinas, Jessica; Schouela, Nicholas; Courchesne, Shannon; Shalev, Uri

    2015-05-01

    Stress is considered to be one of the major triggers to drug relapse, even after prolonged periods of abstinence. In rats, the activation of stress-related brain systems, including corticotropin-releasing factor and norepinephrine, is critical for stress-induced reinstatement of extinguished drug seeking, an animal model for drug relapse. In addition, there are strong indications that activation of the endogenous opioid system is important for the effects of stress on drug seeking. More specifically, activation of the dynorphin/kappa opioid receptor (KOR) system is critically involved in the reinstatement of cocaine seeking following exposure to stressors, such as footshock, forced swimming or social stress. However, studies on the role of the dynorphin/KOR system in stress-induced reinstatement of heroin seeking are scarce. Here, rats were trained to self-administer heroin (0.1 mg/kg/infusion) for 10 days. Drug seeking was then extinguished and the rats were tested for acute (21 hours) food deprivation-induced reinstatement of heroin seeking. In two separate experiments, rats were injected with the mu-opioid receptor (MOR) antagonist, naltrexone (0.0, 1.0, 10.0 mg/kg; s.c.) or the KOR antagonist, norBNI (0.0, 1.0, 10.0 mg/kg; i.p.) before the reinstatement test. Naltrexone treatment did not affect stress-induced reinstatement. In contrast, treatment with norBNI dose-dependently attenuated food deprivation-induced reinstatement of heroin seeking. These results support the hypothesis that activation of KOR, but not MOR, is critically involved in stress-induced reinstatement of drug seeking. PMID:24725195

  2. Reversed-phase liquid chromatographic purification and isolation of a radio-iodinated selective probe for mu opioid receptors in the brain.

    PubMed

    Miller, M M; Gould, B E; Joshi, D; Bennett, H P; James, S; Billiar, R B

    1992-02-01

    A Guard-PAK precolumn system was used for the reversed-phase liquid chromatography purification of a small, synthetic radiolabeled opioid peptide, FK 33-824 (D-Ala2, methyl-phe4, Met (O)ol5 enkephalin) (FK). This procedure involves trace enrichment of iodinated peptide onto the precolumn while iodination reagents are not retained. Radioactive contamination of high-performance liquid chromatography columns and injectors is thus avoided. Precolumn chromatography has sufficient resolving power to separate not only labeled from unlabeled peptide but also mono- from di-iodinated peptide. Purified 125I-labeled FK (estimated specific activity 85.9-153.7 Ci/mmol) showed high specific binding to mouse corpus striatum, neocortex, cingulate cortex, nucleus accumbens septi, diagonal band of Broca, nucleus medialis septi, area preopticus magnocellularis, and the nucleus of the caudate/putamen. Radioligand binding was inhibited by both antagonists (naloxone and naltrexone); and agonists D-Ala2, N-methyl-phe4, gly-ol5-enkephalin [DAGO]; FK; and beta-endorphin at all concentrations tested (1 x 10(-8) to 1 x 10(-4) M). Adrenocorticotropin hormone (ACTH) did not block ligand binding at any concentration tested. Distribution of mu opioid receptors was analyzed by light microscopic autoradiography. Sections incubated with 125I-labeled FK in the presence of agonists and antagonists demonstrated decreasing ligand binding with increasing doses of competitor. ACTH did not block ligand binding at any concentration tested. HPLC analyses of ligand which had been iodinated 1.5 half lives before the date of the experiment demonstrated a single peak similar to that of freshly iodinated ligand. Similar binding kinetics and autoradiographic labeling patterns were observed as compared to those obtained with freshly iodinated peptide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1314320

  3. Sex and age-dependent effects of a maternal junk food diet on the mu-opioid receptor in rat offspring.

    PubMed

    Gugusheff, Jessica R; Bae, Sung Eun; Rao, Alexandra; Clarke, Iain J; Poston, Lucilla; Taylor, Paul D; Coen, Clive W; Muhlhausler, Beverly S

    2016-03-15

    Perinatal junk food exposure increases the preference for palatable diets in juvenile and adult rat offspring. Previous studies have implicated reduced sensitivity of the opioid pathway in the programming of food preferences; however it is not known when during development these changes in opioid signalling first emerge. This study aimed to determine the impact of a maternal junk food (JF) diet on mu-opioid receptor (MuR) expression and ligand binding in two key regions of the reward pathway, the nucleus accumbens (NAc) and the ventral tegmental area (VTA) in rats during the early suckling (postnatal day (PND) 1 and 7) and late suckling/early post-weaning (PND 21 and 28) periods. Female rats were fed either a JF or a control diet for two weeks prior to mating and throughout pregnancy and lactation. MuR expression in the VTA was significantly reduced in female JF offspring on PND 21 and 28 (by 32% and 57% respectively, P<0.05), but not at earlier time points (PND 1 and 7). MuR ligand binding was also reduced (by 22%, P<0.05) in the VTA of female JF offspring on PND 28. No effects of perinatal junk food exposure on MuR mRNA expression or binding were detected at these time points in male offspring. These findings provide evidence that the opioid signalling system is a target of developmental programming by the end of the third postnatal week in females, but not in males. PMID:26718219

  4. Buprenorphine maintenance and mu-opioid receptor availability in the treatment of opioid use disorder: implications for clinical use and policy

    PubMed Central

    Greenwald, Mark K.; Comer, Sandra D.; Fiellin, David A.

    2014-01-01

    Background Sublingual formulations of buprenorphine (BUP) and BUP/naloxone have well-established pharmacokinetic and pharmacodynamic profiles, and are safe and effective for treating opioid use disorder. Since approvals of these formulations, their clinical use has increased. Yet, questions have arisen as to how BUP binding to mu-opioid receptors (μORs), the neurobiological target for this medication, relate to its clinical application. BUP produces dose- and time-related alterations of μOR availability but some clinicians express concern about whether doses higher than those needed to prevent opioid withdrawal symptoms are warranted, and policymakers consider limiting reimbursement for certain BUP dosing regimens. Methods We review scientific data concerning BUP-induced changes in μOR availability and their relationship to clinical efficacy. Results Withdrawal suppression appears to require ≤50% μOR availability, associated with BUP trough plasma concentrations ≥1 ng/mL; for most patients, this may require single daily BUP doses of 4-mg to defend against trough levels, or lower divided doses. Blockade of the reinforcing and subjective effects of typical doses of abused opioids require <20% μOR availability, associated with BUP trough plasma concentrations ≥3 ng/mL; for most individuals, this may require single daily BUP doses >16-mg, or lower divided doses. For individuals attempting to surmount this blockade with higher-than-usual doses of abused opioids, even larger BUP doses and <10% μOR availability would be required. Conclusion For these reasons, and given the complexities of studies on this issue and comorbid problems, we conclude that fixed, arbitrary limits on BUP doses in clinical care or limits on reimbursement for this care are unwarranted. PMID:25179217

  5. Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin

    PubMed Central

    Zhang, Yong; Landthaler, Markus; Schlussman, Stefan D.; Yuferov, Vadim; Ho, Ann; Tuschl, Thomas; Kreek, Mary Jeanne

    2014-01-01

    Mu opioid receptors (MOP-r) play an important role in the rewarding and locomotor stimulatory effects of heroin. The aim of the current study was to determine whether infusion of small interfering RNAs (siRNA) targeting MOP-r into the midbrain could knock down MOP-r mRNA and affect heroin-induced locomotor activity or heroin-induced conditioned place preference. Ten week old male C57BL/6J mice were surgically implanted bilaterally with guide cannulae directed between the substantia nigra and ventral tegmental area. After 4 days recovery, mice were infused bilaterally with siRNAs that target the MOP-r (2mM × 0.75 μl/side/day for 3 days) or control siRNA. Seven days after the last infusion, a procedure for conditioned place preference was begun with four heroin (3mg/kg i.p.) administration sessions alternating with four saline sessions. While heroin induced an increase in locomotor activity in all groups, siRNAs targeting specific regions of MOP-r significantly attenuated this effect. Of particular interest, mice infused with specific siRNAs targeting the MOP-r failed to develop and express conditioned place preference to heroin, or showed a significantly attenuated preference. These alterations in reward related behaviors are likely due to the reduction in MOP-r mRNA and protein, shown in separate studies by in situ hybridization and autoradiography using the same MOP-r- siRNA infusions. Taken together, these studies demonstrate the utility of siRNA in the neurobiological study of specific components of the reward system and should contribute to the study of other complex behaviors. PMID:18938225

  6. C7β-Methyl Analogues of the Orvinols: The Discovery of Kappa Opioid Antagonists with Nociceptin/Orphanin FQ Peptide (NOP) Receptor Partial Agonism and Low, or Zero, Efficacy at Mu Opioid Receptors

    PubMed Central

    2015-01-01

    Buprenorphine is a successful analgesic and treatment for opioid abuse, with both activities relying on its partial agonist activity at mu opioid receptors. However, there is substantial interest in its activities at the kappa opioid and nociceptin/orphanin FQ peptide receptors. This has led to an interest in developing compounds with a buprenorphine-like pharmacological profile but with lower efficacy at mu opioid receptors. The present article describes aryl ring analogues of buprenorphine in which the standard C20-methyl group has been moved to the C7β position, resulting in ligands with the desired profile. In particular, moving the methyl group has resulted in far more robust kappa opioid antagonist activity than seen in the standard orvinol series. Of the compounds synthesized, a number, including 15a, have a profile of interest for the development of drug abuse relapse prevention therapies or antidepressants and others (e.g., 8c), as analgesics with a reduced side-effect profile. PMID:25898137

  7. C7β-methyl analogues of the orvinols: the discovery of kappa opioid antagonists with nociceptin/orphanin FQ peptide (NOP) receptor partial agonism and low, or zero, efficacy at mu opioid receptors.

    PubMed

    Cueva, Juan Pablo; Roche, Christopher; Ostovar, Mehrnoosh; Kumar, Vinod; Clark, Mary J; Hillhouse, Todd M; Lewis, John W; Traynor, John R; Husbands, Stephen M

    2015-05-28

    Buprenorphine is a successful analgesic and treatment for opioid abuse, with both activities relying on its partial agonist activity at mu opioid receptors. However, there is substantial interest in its activities at the kappa opioid and nociceptin/orphanin FQ peptide receptors. This has led to an interest in developing compounds with a buprenorphine-like pharmacological profile but with lower efficacy at mu opioid receptors. The present article describes aryl ring analogues of buprenorphine in which the standard C20-methyl group has been moved to the C7β position, resulting in ligands with the desired profile. In particular, moving the methyl group has resulted in far more robust kappa opioid antagonist activity than seen in the standard orvinol series. Of the compounds synthesized, a number, including 15a, have a profile of interest for the development of drug abuse relapse prevention therapies or antidepressants and others (e.g., 8c), as analgesics with a reduced side-effect profile. PMID:25898137

  8. The Effect of the [mu]-Opioid Receptor Antagonist Naloxone on Extinction of Conditioned Fear in the Developing Rat

    ERIC Educational Resources Information Center

    Kim, Jee Hyun; Richardson, Rick

    2009-01-01

    Several recent studies report that neurotransmitters that are critically involved in extinction in adult rats are not important for extinction in young rats. Specifically, pretest injection of the [gamma]-aminobutryic acid (GABA) receptor inverse agonist FG7142 has no effect on extinction in postnatal day (P)17 rats, although it reverses…

  9. Induction of hyperphagia and carbohydrate intake by mu-opioid receptor stimulation in circumscribed regions of frontal cortex

    PubMed Central

    Mena, Jesus D.; Sadeghian, Ken; Baldo, Brian A.

    2011-01-01

    Frontal cortical regions are activated by food-associated stimuli, and this activation appears to be dysregulated in individuals with eating disorders. Nevertheless, frontal control of basic unconditioned feeding responses remains poorly understood. Here we show that hyperphagia can be driven by μ-opioid receptor stimulation in restricted regions of ventral medial prefrontal cortex (vmPFC) and orbitofrontal cortex. In both ad libitum-fed and food-restricted male Sprague-Dawley rats, bilateral infusions of the μ-opioid agonist, DAMGO, markedly increased intake of standard rat chow. When given a choice between palatable fat- versus carbohydrate enriched test diets, intra-vmPFC DAMGO infusions selectively increased carbohydrate intake, even in rats with a baseline fat preference. Rats also exhibited motor hyperactivity characterized by rapid switching between brief bouts of investigatory and ingestive behaviors. Intra-vmPFC DAMGO affected neither water intake nor non-specific oral behavior. Similar DAMGO infusions into neighboring areas of lateral orbital or anterior motor cortex had minimal effects on feeding. Neither stimulation of vmPFC-localized delta-opioid, kappa-opioid, dopaminergic, serotonergic, or noradrenergic receptors, nor antagonism of D1, 5HT1A, or alpha- or beta-adrenoceptors, reproduced the profile of DAMGO effects. Muscimol-mediated inactivation of the vmPFC, and intra-vmPFC stimulation of κ-opioid receptors or blockade of 5HT2A receptors, suppressed motor activity and increased feeding bout duration-a profile opposite to that seen with DAMGO. Hence, μ-opioid-induced hyperphagia and carbohydrate intake can be elicited with remarkable pharmacological and behavioral specificity from discrete subterritories of the frontal cortex. These findings may have implications for understanding affect-driven feeding and loss of restraint in eating disorders. PMID:21368037

  10. Effects of the Mu Opioid Receptor Polymorphism (OPRM1 A118G) on Pain Regulation, Placebo Effects and Associated Personality Trait Measures

    PubMed Central

    Peciña, Marta; Love, Tiffany; Stohler, Christian S; Goldman, David; Zubieta, Jon-Kar

    2015-01-01

    Mu-opioid receptors (MOPRs) are critically involved in the modulation of pain and analgesia, and represent a candidate mechanism for the development of biomarkers of pain conditions and their responses to treatment. To further understand the human implications of genetic variation within the opioid system in pain and opioid-mediated placebo responses, we investigated the association between the functional single-nucleotide polymorphism (SNP) in the μ-opioid receptor gene (OPRM1), A118G, and psychophysical responses, personality traits, and neurotransmitter systems (dopamine (DA), opioid) related to pain and placebo analgesia. OPRM1 G carriers, compared with AA homozygotes, showed an overall reduction of baseline μ-opioid receptor availability in regions implicated in pain and affective regulation. In response to a sustained painful stimulus, we found no effect of A118G on pain-induced endogenous opioid release. Instead, AA homozygotes showed a blunted DA response in the nucleus accumbens (NAc) in response to the pain challenge. After placebo administration, G carriers showed more pronounced mood disturbances and lower placebo-induced μ-opioid system activation in the anterior insula (aINS), the amygdala (AMY), the NAc, the thalamus (THA), and the brainstem, as well as lower levels of DA D2/3 activation in the NAc. At a trait level, G carriers reported higher NEO-Neuroticism scores; a personality trait previously associated with increased pain and lower placebo responses, which were negatively correlated with baseline μ-opioid receptor availability in the aINS and subgenual anterior cingulate cortex (sgACC). Our results demonstrate that the A118G OPRM1 polymorphism contributes to interindividual variations in the function of neurotransmitters responsive to pain (endogenous opioid and dopamine), as well as their regulation through cognitive-emotional influences in the context of therapeutic expectations, the so-called placebo effect. These effects are relevant to

  11. Low frequency genetic variants in the mu-opioid receptor (OPRM1) affect risk for addiction to heroin and cocaine

    PubMed Central

    Clarke, Toni-Kim; Crist, Richard C.; Kampman, Kyle M.; Dackis, Charles A.; Pettinati, Helen M.; O’Brien, Charles P.; Oslin, David W.; Ferraro, Thomas N.; Lohoff, Falk W.; Berrettini, Wade H.

    2013-01-01

    The μ-opioid receptor (MOR) binds exogenous and endogenous opioids and is known to mediate the rewarding effects of drugs of abuse. Numerous genetic studies have sought to identify common genetic variation in the gene encoding MOR (OPRM1) that affects risk for drug addiction. The purpose of this study was to examine the contribution of rare coding variants in OPRM1 to the risk for addiction. Rare and low frequency variants were selected using the National Heart Lung and Blood Institute –Exome Sequencing Project (NHLBI-ESP) database, which has screened the exomes of over 6500 individuals. Two SNPs (rs62638690 and rs17174794) were selected for genotyping in 1377 European American individuals addicted to heroin and/or cocaine. Two different SNPs (rs1799971 and rs17174801) were genotyped in 1238 African American individuals addicted to heroin and/or cocaine. Using the minor allele frequencies from the NHLBI-ESP dataset as a comparison group, case-control association analyses were performed. Results revealed an association between rs62638690 and cocaine and heroin addiction in European Americans (p=0.02; 95% C.I. 0.47 [0.24–0.92]). This study suggests a potential role for rare OPRM1 variants in addiction disorders and highlights an area worthy of future study. PMID:23454283

  12. mu-Opioid receptor stimulation in the nucleus accumbens elevates fatty tastant intake by increasing palatability and suppressing satiety signals.

    PubMed

    Katsuura, Yoshihiro; Heckmann, Jennifer A; Taha, Sharif A

    2011-07-01

    Infusion of a μ-opioid receptor (MOR) agonist into the nucleus accumbens (NAcc) drives voracious food intake, an effect hypothesized to occur through increased tastant palatability. While intake of many palatable foods is elevated by MOR stimulation, this manipulation has a preferential effect on fatty food ingestion. Consumption of high-fat foods is increased by NAcc MOR stimulation even in rats that prefer a carbohydrate-rich alternative under baseline conditions. This suggests that NAcc MOR stimulation may not simply potentiate palatability signals and raises the possibility that mechanisms mediating fat intake may be distinct from those underlying intake of other tastants. The present study was conducted to investigate the physiological mechanisms underlying the effects of NAcc MOR stimulation on fatty food intake. In experiment 1, we analyzed lick microstructure in rats ingesting Intralipid to identify the changes underlying feeding induced by infusion of a MOR-specific agonist into the NAcc. MOR stimulation in the NAcc core, but not shell, increased burst duration and first-minute licks, while simultaneously increasing the rate and duration of Intralipid ingestion. These results suggest that MOR activation in the core increases Intralipid palatability and attenuates inhibitory postingestive feedback. In experiment 2, we measured the effects of MOR stimulation in the NAcc core on consumption of nonnutritive olestra. A MOR-specific agonist dose dependently increased olestra intake, demonstrating that caloric signaling is not required for hyperphagia induced by NAcc MOR stimulation. Feeding induced by drug infusion in both experiments 1 and 2 was blocked by a MOR antagonist. In experiment 3, we determined whether MOR activation in the NAcc core could attenuate satiety-related signaling caused by infusion of the melanocortin agonist MTII into the third ventricle. Suppression of intake caused by MTII was reversed by MOR stimulation. Together, our results suggest

  13. Nitric oxide and histone deacetylases modulate cocaine-induced mu-opioid receptor levels in PC12 cells

    PubMed Central

    2012-01-01

    Background Cocaine exposure has been reported to alter central μ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be involved in this action of cocaine. Methods To assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin. Results Both cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression. Conclusions In the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine’s action on MOR expression in neurons. PMID:23079001

  14. Exposure to morphine-associated cues increases mu opioid receptor mRNA expression in the nucleus accumbens of Wistar Kyoto rats.

    PubMed

    Dennis, Torry S; Beck, Kevin D; Cominski, Tara P; Bobzean, Samara A M; Kuzhikandathil, Eldo V; Servatius, Richard J; Perrotti, Linda I

    2016-10-15

    The Wistar-Kyoto (WKY) rat has been proposed as a model of anxiety vulnerability as it exhibits pronounced behavioral inhibition, passive avoidance, exaggerated startle response, enhanced HPA-axis activation, and active avoidance that is resistant to extinction. Accumulating evidence suggests that WKY rats respond differently to rewarding stimuli when compared to outbred strains of rat. Conditioned responding to drug-associated cues is linked with alterations in the activation of mu opioid receptors (MOR) and kappa opioid receptors (KOR) in the nucleus accumbens (NAc). Furthermore, alterations in KOR expression/activation in the NAc of WKY rats are implicated in the regulation of some of the components that make up the unique behavioral phenotype of this strain. The purpose of this study was to extend upon previous work from our laboratory by investigating conditioned morphine reward in adult male WKY and SD rats, and to examine levels of KOR mRNA and MOR mRNA in the NAc at baseline and after acquisition of morphine CPP. Our results demonstrate that SD rats displayed morphine-induced CPP to each of the six doses of morphine tested (0.5, 1.25, 2.5, 5, 7.5, or 10mg/kg). Interestingly, WKY rats demonstrated CPP for only the 1.25, 2.5, and 5mg/kg doses, yet no preference at the lowest (0.5mg/kg) or highest (7.5 and 10mg/kg) doses. qPCR analysis of MOR and KOR in the NAc revealed no strain differences in basal levels of MOR, but higher levels of KOR in WKY rats compared to those of SD rats. Interestingly, after completion of the CPP task, WKY rats had overall higher levels of NAc MOR mRNA compared to SD rats; the initial basal differences in NAc KOR levels persisted without change due to CPP in either strain. These results demonstrate that the WKY rat exhibits a unique pattern of behavioral responding to morphine and implicates differences in NAc KOR signaling as a potential source of aversion to higher doses of morphine. Additionally, the CPP-induced upregulation of

  15. The effects of alcohol on the pharmacokinetics and pharmacodynamics of the selective mu-opioid receptor antagonist GSK1521498 in healthy subjects

    PubMed Central

    Ziauddeen, Hisham; Nathan, Pradeep J; Dodds, Chris; Maltby, Kay; Miller, Sam R; Waterworth, Dawn; Song, Kijoung; Warren, Liling; Hosking, Louise; Zucchetto, Mauro; Bush, Mark; Johnson, Lakshmi Vasist; Sarai, Bhopinder; Mogg, Karin; Bradley, Brendan P; Richards, Duncan B; Fletcher, Paul C; Bullmore, Edward T

    2013-01-01

    The mu-opioid system has a key role in hedonic and motivational processes critical to substance addiction. However, existing mu-opioid antagonists have had limited success as anti-addiction treatments. GSK1521498 is a selective and potent mu-opioid antagonist being developed for the treatment of overeating and substance addictions. In this study, 28 healthy participants were administered single doses of GSK1521498 20 mg, ethanol 0.5 g/kg body weight, or both in combination, in a double blind placebo controlled four-way crossover design. The primary objective was to determine the risk of significant adverse pharmacodynamic and pharmacokinetic (PK) interactions. The effects of GSK1521498 on hedonic and consummatory responses to alcohol and the attentional processing of alcohol-related stimuli, and their modulation by the OPRM1 A118G polymorphism were also explored. GSK1521498 20 mg was well tolerated alone and in combination with ethanol. There were mild transient effects of GSK1521498 on alertness and mood that were greater when it was combined with ethanol. These effects were not of clinical significance. There were no effects of GSK1521498 on reaction time, hedonic or consummatory responses. These findings provide encouraging safety and PK data to support continued development of GSK1521498 for the treatment of alcohol addiction. PMID:23934621

  16. Prohormone convertase 2 (PC2) null mice have increased mu opioid receptor levels accompanied by altered morphine-induced antinociception, tolerance and dependence.

    PubMed

    Lutfy, K; Parikh, D; Lee, D L; Liu, Y; Ferrini, M G; Hamid, A; Friedman, T C

    2016-08-01

    Chronic morphine treatment increases the levels of prohormone convertase 2 (PC2) in brain regions involved in nociception, tolerance and dependence. Thus, we tested if PC2 null mice exhibit altered morphine-induced antinociception, tolerance and dependence. PC2 null mice and their wild-type controls were tested for baseline hot plate latency, injected with morphine (1.25-10mg/kg) and tested for antinociception 30min later. For tolerance studies, mice were tested in the hot plate test before and 30min following morphine (5mg/kg) on day 1. Mice then received an additional dose so that the final dose of morphine was 10mg/kg on this day. On days 2-4, mice received additional doses of morphine (20, 40 and 80mg/kg on days 1, 2, 3, and 4, respectively). On day 5, mice were tested in the hot plate test before and 30min following morphine (5mg/kg). For withdrawal studies, mice were treated with the escalating doses of morphine (10, 20, 40 and 80mg/kg) for 4days, implanted with a morphine pellet on day 5 and 3 days later injected with naloxone (1mg/kg) and signs of withdrawal were recorded. Morphine dose-dependently induced antinociception and the magnitude of this response was greater in PC2 null mice. Tolerance to morphine was observed in wild-type mice and this phenomenon was blunted in PC2 null mice. Withdrawal signs were also reduced in PC2 null mice. Immunohistochemical studies showed up-regulation of the mu opioid receptor (MOP) protein expression in the periaqueductal gray area, ventral tegmental area, lateral hypothalamus, medial hypothalamus, nucleus accumbens, and somatosensory cortex in PC2 null mice. Likewise, naloxone specific binding was increased in the brains of these mice compared to their wild-type controls. The results suggest that the PC2-derived peptides may play a functional role in morphine-induced antinociception, tolerance and dependence. Alternatively, lack of opioid peptides led to up-regulation of the MOP and altered morphine

  17. REDUCED EXPRESSION OF THE MU OPIOID RECEPTOR IN SOME, BUT NOT ALL, BRAIN REGIONS IN MICE WITH Oprm1 A112G

    PubMed Central

    WANG, Y.-J.; HUANG, P.; UNG, A.; BLENDY, J. A.; LIU-CHEN, L.-Y.

    2013-01-01

    OPRM1 A118G is a common single nucleotide polymorphism (SNP) in the coding region of the human mu opioid receptor (MOPR) gene OPRM1. This SNP is associated with higher morphine doses required for postoperative analgesia as well as a variety of drug addiction phenotypes. A mouse model possessing the equivalent substitution (A112G) in the Oprm1 gene was generated to facilitate mechanistic studies. Mice homozygous for the G112 allele (G/G) displayed lower antinociception to morphine compared with those homozygous for A112 allele (A/A), similar to humans, suggesting that the mice are a good model to further characterize underlying factors contributing to phenotypes associated with this SNP. Here, we compared [3H]DAMGO binding to the MOPR in the brains of A/A and G/G mice using quantitative in vitro autoradiography. A/A mice exhibited higher [3H]DAMGO binding than G/G in the cingulate, motor, and insular cortices, nucleus accumbens core and shell, hypothalamus, thalamus, amygdala, periaqueductal gray, superficial gray of superior colliculus, and ventral tegmental area. No genotype differences were observed in somatosensory cortex, caudate putamen, and hippocampus. When males and females were examined separately, A/A mice showed higher [3H]DAMGO binding than G/G mice in more brain regions in males than in females. Radioligand binding using brain membranes also showed higher [3H]DAMGO binding in the cortex and thalamus in A/A mice than G/G mice but no genotype differences in the caudate putamen or hippocampus. Thus, the A112G SNP is associated with reduced MOPR expression in some, but not all, brain regions, and appears to have some sex differences. The elevated MOPR expression in periaqueductal gray and thalamus in A/A mice are consistent with their higher antinociceptive responses to morphine. The higher MOPR levels in nucleus accumbens and/or ventral tegmental area of A/A mice is consistent with the higher morphine-induced hyperactivity and locomotor sensitization

  18. Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors.

    PubMed

    Perret, Bénédicte G; Wagner, Renaud; Lecat, Sandra; Brillet, Karl; Rabut, Gwénaël; Bucher, Bernard; Pattus, Franc

    2003-09-01

    The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken. PMID:12963349

  19. General, kappa, delta and mu opioid receptor antagonists mediate feeding elicited by the GABA-B agonist baclofen in the ventral tegmental area and nucleus accumbens shell in rats: reciprocal and regional interactions.

    PubMed

    Miner, Patricia; Shimonova, Lyudmila; Khaimov, Arthur; Borukhova, Yaffa; Ilyayeva, Ester; Ranaldi, Robert; Bodnar, Richard J

    2012-03-14

    Food intake is significantly increased following administration of agonists of GABA and opioid receptors into the nucleus accumbens shell (NACs) and ventral tegmental area (VTA). GABA-A or GABA-B receptor antagonist pretreatment within the VTA or NACs differentially affects mu-opioid agonist-induced feeding elicited from the same site. Correspondingly, general or selective opioid receptor antagonist pretreatment within the VTA or NACs differentially affects GABA agonist-induced feeding elicited from the same site. Regional interactions have been evaluated in feeding studies by administering antagonists in one site prior to agonist administration in a second site. Thus, opioid antagonist-opioid agonist and GABA antagonist-GABA agonist feeding interactions have been identified between the VTA and NACs. However, pretreatment with GABA-A or GABA-B receptor antagonists in the VTA failed to affect mu opioid agonist-induced feeding elicited from the NACs, and correspondingly, these antagonists administered in the NACs failed to affect mu opioid-induced feeding elicited from the VTA. To evaluate whether regional and reciprocal VTA and NACs feeding interactions occur for opioid receptor modulation of GABA agonist-mediated feeding, the present study examined whether feeding elicited by the GABA-B agonist, baclofen microinjected into the NACs was dose-dependently blocked by pretreatment with general (naltrexone: NTX), mu (beta-funaltrexamine: BFNA), kappa (nor-binaltorphamine: NBNI) or delta (naltrindole: NTI) opioid antagonists in the VTA, and correspondingly, whether VTA baclofen-induced feeding was dose-dependently blocked by NACs pretreatment with NTX, BFNA, NBNI or NTI in rats. Bilateral pairs of cannulae aimed at the VTA and NACs were stereotaxically implanted in rats, and their food intakes were assessed following vehicle and baclofen (200 ng) in each site. Baclofen produced similar magnitudes of increased food intake following VTA and NACs treatment. Baclofen

  20. Changes in D1 but not D2 dopamine or mu-opioid receptor expression in limbic and motor structures after lateral hypothalamus electrical self-stimulation: A quantitative autoradiographic study.

    PubMed

    Simon, Maria J; Higuera-Matas, A; Roura-Martinez, D; Ucha, M; Santos-Toscano, R; Garcia-Lecumberri, C; Ambrosio, E; Puerto, A

    2016-01-01

    Intracranial self-stimulation (ICSS) of the lateral hypothalamus (LH) is involved in the activation of neuroanatomical systems that are also associated with the processing of natural and other artificial rewarding stimuli. Specific components of this behavior (hedonic impact, learning, and motor behavior) may involve changes in different neurotransmitters, such as dopamine and opioids. In this study, quantitative autoradiography was used to examine changes in mu-opioid and D1/D2-dopamine receptor expression in various anatomical regions related to the motor and mesolimbic reward systems after intracranial self-stimulation of the LH. Results of the behavioral procedure and subsequent radiochemical assays show selective changes in D1 but not D2 or mu receptors in Accumbens-Shell, Ventral Pallidum, Caudate-Putamen, and Medial Globus Pallidus. These findings are discussed in relation to the different psychobiological components of the appetitive motivational system, identifying some dissociation among them, particularly with respect to the involvement of the D1-dopamine subsystem (but not D2 or mu receptors) in goal-directed behaviors. PMID:26656274

  1. Pretreatment of rats with the irreversible mu-receptor antagonist, beta-FNA, fails to prevent naltrexone-induced upregulation of mu-opioid receptors.

    PubMed

    Rothman, R B; Long, J B; Bykov, V; Jacobson, A E; Rice, K C; Holaday, J W

    1990-09-01

    This study examined the effect of beta-funaltrexamine (beta-FNA), an irreversible mu-receptor antagonist, on naltrexone-induced upregulation of mu-(mu cx + mu nex) and delta nex-opioid receptors. [The subscripts 'cx' and 'nex' denote binding sites 'in' (cx) and 'not in' (nex) the opioid receptor complex.] Rats were treated according to the following protocol. Two naltrexone or two placebo pellets were implanted subcutaneously in a nylon mesh on day 1. and were removed intact on day 8. Rats were given either saline or 20 nmol of beta-FNA in 10 microliters of saline (i.c.v.) on days 1, 3, 5 and 6, 60 min prior to implantation of the pellet. On day 9 frozen lysed-P2 membranes were prepared for assay of mu binding sites. In other experiments, membranes were depleted of mu-receptors by pretreatment with the site-directed acylating agent 2-(4-ethoxybenzyl)-l-diethylaminoethyl-5-isothiocyanatobenzimid azole.HCl (BIT) for assay of delta nex binding sites, using [3H] [D-ala2, D-leu5]enkephalin. The results demonstrated that beta-FNA did not upregulate the mu binding sites and also did not prevent naltrexone-induced upregulation of mu binding sites. Both beta-FNA and naltrexone increased the Bmax of delta nex binding sites and their effects were additive. These data suggest that the mechanism(s) responsible for antagonist-induced upregulation of opioid receptors are more complex than previously appreciated. PMID:1963479

  2. Synthesis and evaluation of aryl-naloxamide opiate analgesics targeting truncated exon 11-associated mu opioid receptor (MOR-1) splice variants

    PubMed Central

    Majumdar, Susruta; Subrath, Joan; Le Rouzic, Valerie; Polikar, Lisa; Burgman, Maxim; Nagakura, Kuni; Ocampo, Julie; Haselton, Nathan; Pasternak, Anna R.; Grinnell, Steven; Pan, Ying-Xian; Pasternak, Gavril W.

    2012-01-01

    3-Iodobenzoylnaltrexamide 1 (IBNtxA) is a potent analgesic acting through a novel receptor target that lack many side-effects of traditional opiates composed, in part, of exon 11-associated truncated six transmembrane domain MOR-1 (6TM/E11) splice variants. To better understand the SAR of this drug target, a number of 4,5-epoxymorphinan analogs were synthesized. Results show the importance of a free 3-phenolic group, a phenyl ring at the 6 position, an iodine at the 3′ or 4′ position of the phenyl ring and an N-allyl or c-propylmethyl group to maintain high 6TM/E11 affinity and activity. 3-Iodobenzoylnaloxamide 15 (IBNalA) with a N-allyl group displayed lower delta opioid receptor affinity than its naltrexamine analog, was 10-fold more potent an analgesic than morphine, elicited no respiratory depression or physical dependence and only limited inhibition of gastrointestinal transit. Thus, the aryl-naloxamide scaffold can generate a potent analgesic acting through the 6TM/E11 sites with advantageous side-effect profile and greater selectivity. PMID:22734622

  3. /sup 3/H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((/sup 3/H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

    SciTech Connect

    Hawkins, K.N.; Knapp, R.J.; Lui, G.K.; Gulya, K.; Kazmierski, W.; Wan, Y.P.; Pelton, J.T.; Hruby, V.J.; Yamamura, H.I.

    1989-01-01

    The cyclic, conformationally restricted octapeptide (3H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of (3H)CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. (3H)CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on (3H)CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide (3H)-(D-Pen2,D-Pen5)enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific (3H)CTOP binding was inhibited by a large number of opioid or opiate ligands.

  4. Behavioral and Cellular Pharmacology Characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan (NAQ) as a Mu Opioid Receptor Selective Ligand

    PubMed Central

    Zhang, Yan; Braithwaite, Amanda; Yuan, Yunyun; Streicher, John M.; Bilsky, Edward J.

    2014-01-01

    Mu opioid receptor (MOR) selective antagonists and partial agonists have been used for the treatment of opioid abuse and addiction. Our recent efforts on the identification of MOR antagonists have provided several novel leads displaying interesting pharmacological profiles. Among them, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamido]morphinan (NAQ) showed sub-nanomolar binding affinity to the MOR with significant selectivity over the delta opioid receptor (DOR) and the kappa opioid receptor (KOR). Its central nervous system penetration capacity together with marginal agonism in the MOR–GTPγS binding assay made it a very interesting molecule for developing novel opioid abuse and addiction therapeutic agents. Therefore, further pharmacological characterization was conducted to fully understand its biological profile. At the molecular and cellular level, NAQ not only induced no translocation of β-arrestin2 to the MOR, but also efficaciously antagonized the effect of DAMGO in MOR-βarr2eGFP-U2OS cells in the β-arrestin2 recruitment assay. At the in vivo level, NAQ displayed a potent inhibition of the analgesic effect of morphine in the tail-flick assay (ID50 = 1.19 mg/kg). NAQ (10 mg/kg) also significantly decreased the hyper-locomotion induced by acute morphine without inducing any vertical jumps. Meanwhile NAQ precipitated lesser withdrawal symptoms in morphine dependent mice than naloxone. In conclusion, NAQ may represent a new chemical entity for opioid abuse and addiction treatment. PMID:24815322

  5. Increased mesolimbic cue-reactivity in carriers of the mu-opioid-receptor gene OPRM1 A118G polymorphism predicts drinking outcome: a functional imaging study in alcohol dependent subjects.

    PubMed

    Bach, Patrick; Vollsta Dt-Klein, Sabine; Kirsch, Martina; Hoffmann, Sabine; Jorde, Anne; Frank, Josef; Charlet, Katrin; Beck, Anne; Heinz, Andreas; Walter, Henrik; Sommer, Wolfgang H; Spanagel, Rainer; Rietschel, Marcella; Kiefer, Falk

    2015-08-01

    The endogenous opioid system is involved in the pathophysiology of alcohol-use disorders. Genetic variants of the opioid system alter neural and behavioral responses to alcohol. In particular, a single nucleotide polymorphism rs1799971 (A118G) in the mu-opioid receptor gene (OPRM1) is suggested to modulate alcohol-related phenotypes and neural response in the mesocorticolimbic dopaminergic system. Little is known about the clinical implications of these changes. The current study investigated the relationship of genotype effects on subjective and neural responses to alcohol cues and relapse in a sample of abstinent alcohol-dependent patients. Functional magnetic resonance imaging (fMRI) was used to investigate alcohol cue-reactivity and drinking outcome of 81 abstinent alcohol-dependent patients. G-allele carriers displayed increased fMRI cue-reactivity in the left dorsal striatum and bilateral insulae. Neural responses to alcohol cues in these brain regions correlated positively with subjective craving for alcohol and positive expectations of alcohol׳s effects. Moreover, alcohol cue-reactivity in the left dorsal striatum predicted time to first severe relapse. Current results show that alcohol-dependent G-allele carriers׳ increased cue-reactivity is associated with an increased relapse risk. This suggests that genotype effects on cue-reactivity might link the OPRM1 A118G risk allele with an increased relapse risk that was reported in earlier studies. From a clinical perspective, risk-allele carriers might benefit from treatments, such as neuro-feedback or extinction-based therapy that are suggested to reduce mesolimbic reactivity. PMID:25937240

  6. Knockdown of ventral tegmental area mu-opioid receptors in rats prevents effects of social defeat stress: Implications for amphetamine cross-sensitization, social avoidance, weight regulation and expression of brain-derived neurotrophic factor

    PubMed Central

    Johnston, Caitlin E.; Herschel, Daniel; Lasek, Amy W.; Hammer, Ronald P.; Nikulina, Ella M.

    2014-01-01

    Social defeat stress causes social avoidance and long-lasting cross-sensitization to psychostimulants, both of which are associated with increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). Moreover, social stress upregulates VTA mu-opioid receptor (MOR) mRNA. In the VTA, MOR activation inhibits GABA neurons to disinhibit VTA dopamine neurons, thus providing a role for VTA MORs in the regulation of psychostimulant sensitization. The present study determined the effect of lentivirus-mediated MOR knockdown in the VTA on the consequences of intermittent social defeat stress, a salient and profound stressor in humans and rodents. Social stress exposure induced social avoidance and attenuated weight gain in animals with non-manipulated VTA MORs, but both these effects were prevented by VTA MOR knockdown. Rats with non-manipulated VTA MOR expression exhibited cross-sensitization to amphetamine challenge (1.0 mg/kg, i.p.), evidenced by a significant augmentation of locomotion. By contrast, knockdown of VTA MORs prevented stress-induced cross-sensitization without blunting the locomotor-activating effects of amphetamine. At the time point corresponding to amphetamine challenge, immunohistochemical analysis was performed to examine the effect of stress on VTA BDNF expression. Prior stress exposure increased VTA BDNF expression in rats with non-manipulated VTA MOR expression, while VTA MOR knockdown prevented stress-induced expression of VTA BDNF. Taken together, these results suggest that upregulation of VTA MOR is necessary for the behavioral and biochemical changes induced by social defeat stress. Elucidating VTA MOR regulation of stress effects on the mesolimbic system may provide new therapeutic targets for treating stress-induced vulnerability to substance abuse. PMID:25446676

  7. Opposite effects of delta and mu opioid receptor agonists on the in vitro release of substance P-like material from the rat spinal cord.

    PubMed

    Mauborgne, A; Lutz, O; Legrand, J C; Hamon, M; Cesselin, F

    1987-02-01

    Superfusion of slices from the dorsal half of the lumbar enlargement of rat spinal cord with Krebs-Henseleit medium supplemented with 30 microM bacitracin allowed the collection of substance P-like immunoreactive material (SPLI), which was released at a rate of approximately 10 pg/4 min. Tissue depolarization by an excess of K+ (30-60 mM) or veratridine (50 microM) induced a marked increase in SPLI outflow, provided that Ca2+ was present in the superfusing fluid. K+- or veratridine-induced SPLI overflow could be modulated in opposite directions by mu and delta opioid receptor agonists. Thus, the two preferential mu agonists Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO; 10 microM) and Tyr-D-Ala-Gly-MePhe-Met(O)5-OH (FK-33824; 0.1 microM) enhanced SPLI overflow from depolarized tissues, whereas the selective delta agonists Tyr-D-Thr-Gly-Phe-Leu-Thr (deltakephalin; 3 microM) and [2-D-penicillamine, 5-D-penicillamine]enkephalin (50 microM) reduced it. The effect of DAGO was antagonized by a low concentration (1 microM) of naloxone but not by the selective delta antagonist ICI-154129 (50 microM). In contrast, the latter drug prevented the inhibitory influence of delta agonists on K+-induced SPLI release. Complementary experiments with morphine (10 microM) and [2-D-alanine, 5-D-leucine]enkephalinamide (3 microM), in combination with 1 microM naloxone or 50 microM ICI-154129 for the selective blockade of mu or delta receptors, respectively, confirmed that the stimulation of mu receptors increased, whereas the stimulation of delta receptors reduced, SPLI overflow. The results suggest that, at the spinal level, and antinociceptive action of delta but not mu agonists might involve a presynaptic inhibition of substance P-containing primary afferent fibers. PMID:2432185

  8. Involvement of Mu Opioid Receptor Signaling in the Protective Effect of Opioid against 6-Hydroxydopamine-Induced SH-SY5Y Human Neuroblastoma Cells Apoptosis

    PubMed Central

    Eftekhar-Vaghefi, Shahrzad; Esmaeili-Mahani, Saeed; Elyasi, Leila; Abbasnejad, Mehdi

    2015-01-01

    Introduction: The neuroprotective role of opioid morphine against 6-hydroxydopamine-induced cell death has been demonstrated. However, the exact mechanism(s) underlying such neuroprotection, especially the role of subtype receptors, has not yet been fully clarified. Methods: Here, we investigated the effects of different opioid agonists on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson’s disease. Cell damage was induced by 150 μM 6-OHDA and the cells viability was examined by MTT assay. Intracellular calcium, reactive oxygen species and mitochondrial membrane potential were assessed by fluorescence spectrophotometry method. Immunoblot technique was used to evaluate cytochrome-c and activated caspase-3 as biochemical markers of apoptosis induction. Results: The data showed that 6-OHDA caused significant cell damage, loss of mitochondrial membrane potential and increase in intracellular reactive oxygen species and calcium levels as well as activated caspase-3 and cytochrome-c release. Incubation of SH-SY5Y cells with μ-opioid agonists, morphine and DAMGO, but not with δ-opioid agonist, DADLE, elicited protective effect and reduced biochemical markers of cell damage and death. Discussion: The results suggest that μ-opioid receptors signaling participate in the opioid neuroprotective effects against 6-OHDA-induced neurotoxicity. PMID:26904174

  9. Effects of a mu-opioid receptor agonist (codeine phosphate) on visuo-motor coordination and dynamic visual acuity in man.

    PubMed Central

    Bradley, C M; Nicholson, A N

    1986-01-01

    Effects of codeine (30, 60 and 90 mg) on visuo-motor coordination and dynamic visual acuity, together with critical flicker fusion, digit symbol substitution, complex reaction time and subjective assessments of mood, were studied from 0.75-2.0 h after ingestion by six healthy female adults. The study was double-blind and placebo controlled, and triprolidine (10 mg) was used as the active control. The effect on visuo-motor coordination was limited and was dose related and linear, and performance was altered on visuo-motor coordination with 60 and 90 mg codeine, and on dynamic visual acuity with 90 mg codeine (P less than 0.05). No other effect of codeine was detected. Modulated neuromuscular function is likely to be the common denominator of the changes in performance with codeine, though nausea, but not sedation, may be a contributory factor. It is possible that altered performance with codeine may involve interactions with different receptors than those which lead to sedation. PMID:3024689

  10. Elevated mu-opioid receptor expression in the nucleus of the solitary tract accompanies attenuated withdrawal signs after chronic low dose naltrexone in opiate-dependent rats.

    PubMed

    Van Bockstaele, E J; Rudoy, C; Mannelli, P; Oropeza, V; Qian, Y

    2006-02-15

    We previously described a decrease in withdrawal behaviors in opiate-dependent rats that were chronically treated with very low doses of naltrexone in their drinking water. Attenuated expression of withdrawal behaviors correlated with decreased c-Fos expression and intracellular signal transduction elements [protein kinase A regulatory subunit II (PKA) and phosphorylated cAMP response element binding protein (pCREB)] in brainstem noradrenergic nuclei. In this study, to determine whether similar cellular changes occurred in forebrain nuclei associated with drug reward, expressions of PKA and pCREB were analyzed in the ventral tegmental area, frontal cortex, striatum, and amygdala of opiate-treated rats that received low doses of naltrexone in their drinking water. No significant difference in PKA or pCREB was detected in these regions following drug treatment. To examine further the cellular mechanisms in noradrenergic nuclei that could underlie attenuated withdrawal behaviors following low dose naltrexone administration, the nucleus of the solitary tract (NTS) and locus coeruleus (LC) were examined for opioid receptor (OR) protein expression. Results showed a significant increase in muOR expression in the NTS of morphine-dependent rats that received low doses of naltrexone in their drinking water, and increases in muOR expression were also found to be dose dependent. Protein expression of muOR in the LC and deltaOR in either brain region remained unchanged. In conclusion, our previously reported decreases in c-Fos and PKA expression in the NTS following pretreatment with low doses of naltrexone may be partially explained by a greater inhibition of NTS neurons resulting from increased muOR expression in this region. PMID:16385558

  11. Mu Opioid Mediated Discriminative-Stimulus Effects of Tramadol: An Individual Subjects Analysis

    PubMed Central

    Strickland, Justin C.; Rush, Craig R.; Stoops, William W.

    2015-01-01

    Drug discrimination procedures use dose-dependent generalization, substitution, and pretreatment with selective agonists and antagonists to evaluate receptor systems mediating interoceptive effects of drugs. Despite the extensive use of these techniques in the nonhuman animal literature, few studies have used human subjects. Specifically, human studies have not routinely used antagonist administration as a pharmacological tool to elucidate the mechanisms mediating the discriminative stimulus effects of drugs. This study evaluated the discriminative-stimulus effects of tramadol, an atypical analgesic with monoamine and mu opioid activity. Three human subjects first learned to discriminate 100 mg tramadol from placebo. A range of tramadol doses (25 to 150 mg) and hydromorphone (4 mg) with and without naltrexone pretreatment (50 mg) were then administered to subjects after acquiring the discrimination. Tramadol produced dose-dependent increases in drug-appropriate responding and hydromorphone partially or fully substituted for tramadol in all subjects. These effects were attenuated by naltrexone. Individual subject records indicated a relationship between mu opioid activity (i.e., miosis) and drug discrimination performance. Our findings indicate that mu opioid activity may mediate the discriminative-stimulus effects of tramadol in humans. The correspondence of generalization, substitution, and pretreatment findings with the animal literature supports the neuropharmacological specificity of the drug discrimination procedure. PMID:25664525

  12. (/sup 3/H)(D-Ala2,NMePhe4,Gly-ol5)-enkephalin (mu-opioid) binding in beige-J mice

    SciTech Connect

    Raffa, R.B.; Baldy, W.J. Jr.; Shank, R.P.; Mathiasen, J.R.; Vaught, J.L.

    1988-05-01

    Tritiated (D-Ala2,NMePhe4,Gly-ol5)-enkephalin ((3H)DAGO) was used to examine mu-opioid receptor number and mu-ligand binding in brain synaptic membranes (P2 fraction) from C57BL/6J-bgJ/bgJ (beige-J) mice, a strain with combined deficiencies in immunological function (resembling Chediak-Higashi syndrome) and analgesic response to mu-opioid agonists such as morphine and DAGO. As controls, white mice, beige-J littermates (normally responsive to mu-opioid agonists), and a known mu-deficient strain (CXBK) were also examined. Neither the KD (0.47 to 0.49 nM) nor the Bmax (153 to 168 fmol/mg protein) determined for beige-J mice was significantly different from values determined for littermates or white mice. In contrast, the Bmax of CXBK mice (66 fmol/mg protein) was clearly less than that of the other strains. The analgesic defect of beige-J mice, therefore, is not likely due to an insufficient number of mu-opioid receptors, as it presumably is in CXBK mice. Carbachol (200 micrograms/ml), which partly corrects the analgesic defect of beige-J mice, had no effect on (3H)DAGO binding either acutely in vitro or chronically ex vivo after administration to beige-J mice for three weeks. Hence, the analgesic defect of beige-J mice appears to be due to some defect in the mu-opioid receptor-effector coupling mechanism or to some endogenous substance that inhibits binding of mu-opioid ligands to otherwise functional receptors.

  13. Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS.

    PubMed

    Marrone, Gina F; Grinnell, Steven G; Lu, Zhigang; Rossi, Grace C; Le Rouzic, Valerie; Xu, Jin; Majumdar, Susruta; Pan, Ying-Xian; Pasternak, Gavril W

    2016-03-29

    The clinical management of severe pain depends heavily on opioids acting through mu opioid receptors encoded by the Oprm1 gene, which undergoes extensive alternative splicing. In addition to generating a series of prototypic seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs), Oprm1 also produces a set of truncated splice variants containing only six transmembrane domains (6TM) through which selected opioids such as IBNtxA (3'-iodobenzoyl-6β-naltrexamide) mediate a potent analgesia without many undesirable effects. Although morphine analgesia is independent of these 6TM mu receptor isoforms, we now show that the selective loss of the 6TM variants in a knockout model eliminates the analgesic actions of delta and kappa opioids and of α2-adrenergic compounds, but not cannabinoid, neurotensin, or muscarinic drugs. These observations were confirmed by using antisense paradigms. Despite their role in analgesia, loss of the 6TM variants were not involved with delta opioid-induced seizure activity, aversion to the kappa drug U50, 488H, or α2-mediated hypolocomotion. These observations support the existence of parallel opioid and nonopioid pain modulatory systems and highlight the ability to dissociate unwanted delta, kappa1, and α2 actions from analgesia. PMID:26976581

  14. The competitive N-methyl-D-aspartate receptor antagonist (-)-6-phosphonomethyl-deca-hydroisoquinoline-3-carboxylic acid (LY235959) potentiates the antinociceptive effects of opioids that vary in efficacy at the mu-opioid receptor.

    PubMed

    Allen, Richard M; Granger, Arthur L; Dykstra, Linda A

    2003-11-01

    (-)-6-Phosphonomethyl-deca-hydroisoquinoline-3-carboxylic acid (LY235959) is a competitive N-methyl-D-aspartate receptor antagonist shown to prevent the development of tolerance to the antinociceptive effects of morphine in rodents. Although administration of LY235959 alone generally does not produce antinociception, LY235959 potentiates the antinociceptive effects of morphine in squirrel monkeys. The present study was designed to determine whether LY235959 would potentiate the acute antinociceptive effects of morphine as well those of the opioid receptor agonists l-methadone, levorphanol, butorphanol, and buprenorphine. A squirrel monkey titration procedure was used in which shock (delivered to the tail) increased in intensity every 15 s (0.01-2.0 mA) in 30 increments. Five lever presses during any given 15-s shock period (fixed ratio 5) produced a 15-s shock-free period after which shock resumed at the next lower intensity. Morphine (0.3-3.0 mg/kg i.m.), l-methadone (0.1-0.56 mg/kg i.m.), levorphanol (0.1-1.0 mg/kg i.m.), butorphanol (1.0-10 mg/kg i.m.), and buprenorphine (0.01-0.03 mg/kg i.m.), but not LY235959 (0.1-1.0 mg/kg i.m.), dose and time dependently increased the intensity below which monkeys maintained shock 50% of the time (median shock level, MSL). LY235959 dose dependently potentiated the effect of each opioid agonist on MSL when concurrently administered to monkeys. Although LY235959 potentiated the antinociceptive effect of each opioid examined in a statistically significant manner, LY235959 seemed more potent and effective when combined with higher efficacy opioids. The present data suggest that the N-methyl-D-aspartate antagonist, LY235959, can potentiate the antinociceptive effects of a range of opioid receptor agonists independently of nonspecific motor effects. PMID:12975489

  15. Chronic exercise decreases sensitivity to mu opioids in female rats: correlation with exercise output.

    PubMed

    Smith, Mark A; Lyle, Megan A

    2006-09-01

    Aerobic exercise stimulates the release of endogenous opioid peptides and increases nociceptive (i.e., pain) threshold in a naloxone-reversible manner. During chronic exercise, sensitivity to the antinociceptive effects of morphine and other mu opioids decreases, leading some investigators to propose that exercise may lead to the development of cross-tolerance to exogenously administered opioid agonists. The purpose of the present study was to examine the effects of chronic exercise on sensitivity to mu opioids, and to determine if changes in opioid sensitivity during chronic exercise are correlated with exercise output. Eight female rats were obtained at weaning and housed in standard laboratory cages that did not permit any exercise beyond normal cage ambulation. Following 6 weeks under these conditions, opioids possessing a range of relative efficacies at the mu receptor (morphine, levorphanol, buprenorphine, butorphanol) were examined in a warm-water, tail-withdrawal procedure. Under sedentary conditions, all opioids produced dose-dependent increases in tail-withdrawal latencies, and high levels of antinociception were observed for all drugs. Following these tests, rats were reassigned to exercise conditions and transferred to cages equipped with running wheels. Under these conditions, rats ran an average of 7154 rev/day (7869 m/day), with a range across rats from 4501 to 10,164 rev/day (4951-11,180 m/day). Sensitivity to all four opioids decreased significantly during the exercise period, resulting in 2- to 5-fold decreases in the potency of morphine, levorphanol and buprenorphine, and decreases in the effectiveness of buprenorphine and butorphanol. When rats were returned to sedentary conditions, sensitivity to all four opioids increased significantly and returned to that observed prior to the exercise period. For all drugs, there was a positive correlation between exercise output and changes in opioid sensitivity between sedentary and exercise conditions

  16. Central HIV-1 Tat exposure elevates anxiety and fear conditioned responses of male mice concurrent with altered mu-opioid receptor-mediated G-protein activation and β-arrestin 2 activity in the forebrain.

    PubMed

    Hahn, Yun K; Paris, Jason J; Lichtman, Aron H; Hauser, Kurt F; Sim-Selley, Laura J; Selley, Dana E; Knapp, Pamela E

    2016-08-01

    Co-exposure to opiates and HIV/HIV proteins results in enhanced CNS morphological and behavioral deficits in HIV(+) individuals and in animal models. Opiates with abuse liability, such as heroin and morphine, bind preferentially to and have pharmacological actions through μ-opioid-receptors (MORs). The mechanisms underlying opiate-HIV interactions are not understood. Exposure to the HIV-1 transactivator of transcription (Tat) protein causes neurodegenerative outcomes that parallel many aspects of the human disease. We have also observed that in vivo exposure to Tat results in apparent changes in morphine efficacy, and thus have hypothesized that HIV proteins might alter MOR activation. To test our hypothesis, MOR-mediated G-protein activation was determined in neuroAIDS-relevant forebrain regions of transgenic mice with inducible CNS expression of HIV-1 Tat. G-protein activation was assessed by MOR agonist-stimulated [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPγS) autoradiography in brain sections, and in concentration-effect curves of MOR agonist-stimulated [(35)S]GTPγS binding in membranes isolated from specific brain regions. Comparative studies were done using the MOR-selective agonist DAMGO ([D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin) and a more clinically relevant agonist, morphine. Tat exposure reduced MOR-mediated G-protein activation in an agonist, time, and regionally dependent manner. Levels of the GPCR regulatory protein β-arrestin-2, which is involved in MOR desensitization, were found to be elevated in only one affected brain region, the amygdala; amygdalar β-arrestin-2 also showed a significantly increased association with MOR by co-immunoprecipitation, suggesting decreased availability of MOR. Interestingly, this correlated with changes in anxiety and fear-conditioned extinction, behaviors that have substantial amygdalar input. We propose that HIV-1 Tat alters the intrinsic capacity of MOR to signal in response to agonist binding

  17. Mu-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening.

    PubMed

    Smart, D; Smith, G; Lambert, D G

    1995-01-15

    We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated. PMID:7832776

  18. Mu Opioid Splice Variant MOR-1K Contributes to the Development of Opioid-Induced Hyperalgesia

    PubMed Central

    Oladosu, Folabomi A.; Conrad, Matthew S.; O’Buckley, Sandra C.; Rashid, Naim U.; Slade, Gary D.; Nackley, Andrea G.

    2015-01-01

    Background A subset of the population receiving opioids for the treatment of acute and chronic clinical pain develops a paradoxical increase in pain sensitivity known as opioid-induced hyperalgesia. Given that opioid analgesics are one of few treatments available against clinical pain, it is critical to determine the key molecular mechanisms that drive opioid-induced hyperalgesia in order to reduce its prevalence. Recent evidence implicates a splice variant of the mu opioid receptor known as MOR-1K in the emergence of opioid-induced hyperalgesia. Results from human genetic association and cell signaling studies demonstrate that MOR-1K contributes to decreased opioid analgesic responses and produces increased cellular activity via Gs signaling. Here, we conducted the first study to directly test the role of MOR-1K in opioid-induced hyperalgesia. Methods and Results In order to examine the role of MOR-1K in opioid-induced hyperalgesia, we first assessed pain responses to mechanical and thermal stimuli prior to, during, and following chronic morphine administration. Results show that genetically diverse mouse strains (C57BL/6J, 129S6, and CXB7/ByJ) exhibited different morphine response profiles with corresponding changes in MOR-1K gene expression patterns. The 129S6 mice exhibited an analgesic response correlating to a measured decrease in MOR-1K gene expression levels, while CXB7/ByJ mice exhibited a hyperalgesic response correlating to a measured increase in MOR-1K gene expression levels. Furthermore, knockdown of MOR-1K in CXB7/ByJ mice via chronic intrathecal siRNA administration not only prevented the development of opioid-induced hyperalgesia, but also unmasked morphine analgesia. Conclusions These findings suggest that MOR-1K is likely a necessary contributor to the development of opioid-induced hyperalgesia. With further research, MOR-1K could be exploited as a target for antagonists that reduce or prevent opioid-induced hyperalgesia. PMID:26270813

  19. Aminothiazolomorphinans with Mixed Kappa and Mu Opioid Activitya

    PubMed Central

    Zhang, Tangzhi; Yan, Zhaohua; Sromek, Anna; Knapp, Brian I.; Scrimale, Thomas; Bidlack, Jean M.; Neumeyer, John L.

    2011-01-01

    A series of N-substituted and N′-substituted aminothiazole-derived morphinans (5) were synthesized for expanding the structure-activity relationships of aminothiazolo-morphinans. Although their affinities were somewhat lower than their prototype aminothiazolo-N-cyclopropylmorphinan (3), 3-aminothiazole derivatives of cyclorphan (1) containing a primary amino group displayed high affinity and selectivity at the κ and μ opioid receptors. [35S]GTPγS binding assays showed that the aminothiazolomorphinans were κ agonists with mixed agonist and antagonist activity at the μ opioid receptor. These novel N′-monosubstituted aminothiazole-derived morphinans may be valuable for the development of drug abuse medications. PMID:21351746

  20. μ Opioid Receptor Expression after Morphine Administration Is Regulated by miR-212/132 Cluster

    PubMed Central

    Garcia-Concejo, Adrian; Jimenez-Gonzalez, Ada; Rodríguez, Raquel E.

    2016-01-01

    Since their discovery, miRNAs have emerged as a promising therapeutical approach in the treatment of several diseases, as demonstrated by miR-212 and its relation to addiction. Here we prove that the miR-212/132 cluster can be regulated by morphine, through the activation of mu opioid receptor (Oprm1). The molecular pathways triggered after morphine administration also induce changes in the levels of expression of oprm1. In addition, miR-212/132 cluster is actively repressing the expression of mu opioid receptor by targeting a sequence in the 3’ UTR of its mRNA. These findings suggest that this cluster is closely related to opioid signaling, and function as a post-transcriptional regulator, modulating morphine response in a dose dependent manner. The regulation of miR-212/132 cluster expression is mediated by MAP kinase pathway, CaMKII-CaMKIV and PKA, through the phosphorylation of CREB. Moreover, the regulation of both oprm1 and of the cluster promoter is mediated by MeCP2, acting as a transcriptional repressor on methylated DNA after prolonged morphine administration. This mechanism explains the molecular signaling triggered by morphine as well as the regulation of the expression of the mu opioid receptor mediated by morphine and the implication of miR-212/132 in these processes. PMID:27380026

  1. Fentanyl, an agonist at the mu opioid receptor, depresses pupillary unrest.

    PubMed

    Bokoch, Michael P; Behrends, Matthias; Neice, Andrew; Larson, Merlin D

    2015-05-01

    Pupillary unrest is a chaotic fluctuation in pupil size that is observed in darkness with the onset of drowsiness, and in ambient light. The mechanism of pupillary unrest in darkness as well as in ambient light is unknown but studies suggest that it is caused by fluctuating activity in the Edinger-Westphal (E.W.) nucleus. Neurons in the periaqueductal gray with oscillating firing patterns that are inhibitory to the E.W. nucleus have been described in the cat. We theorized that such oscillating neurons produce pupillary unrest in light and would be depressed by agents, such as opioids, known to depress inhibitory pathways in the midbrain. An infrared pupillometer was used to measure the effect of light on pupillary unrest in eight volunteer subjects, and on 20 patients scheduled for knee arthroscopy who received fentanyl as premedication. Pupillary unrest was quantified through spectral analysis of fast Fourier transforms. Sixteen-second measurements of pupil size at 33 Hz were filtered to eliminate blink artifacts and baseline drift. Pupillary unrest was augmented by excitation of the E.W. nucleus by light and was depressed by 40 ± 20% after the administration of the moderate dose of 1 mcg/kg of fentanyl. Recovery from the drug effect was observed. Based upon the data from this study we propose that pupillary unrest in light originates within oscillating inhibitory neurons that intermittently depress the E. W. nucleus. PMID:25737234

  2. Wheel running reduces high-fat diet intake, preference and mu-opioid agonist stimulated intake

    PubMed Central

    Liang, Nu-Chu; Bello, Nicholas T.; Moran, Timothy H.

    2015-01-01

    The ranges of mechanisms by which exercise affects energy balance remain unclear. One potential mechanism may be that exercise reduces intake and preference for highly palatable, energy dense fatty foods. The current study used a rodent wheel running model to determine whether and how physical activity affects HF diet intake/preference and reward signaling. Experiment 1 examined whether wheel running affected the ability of intracerebroventricular (ICV) µ opioid receptor agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO) to increase HF diet intake. Experiment 2 examined the effects of wheel running on the intake of and preference for a previously preferred HF diet. We also assessed the effects of wheel running and diet choice on mesolimbic dopaminergic and opioidergic gene expression. Experiment 1 revealed that wheel running decreased the ability of ICV DAMGO administration to stimulate HF diet intake. Experiment 2 showed that wheel running suppressed weight gain and reduced intake and preference for a previously preferred HF diet. Furthermore, the mesolimbic gene expression profile of wheel running rats was different from that of their sedentary paired-fed controls but similar to that of sedentary rats with large HF diet consumption. These data suggest that alterations in preference for palatable, energy dense foods play a role in the effects of exercise on energy homeostasis. The gene expression results also suggest that the hedonic effects of exercise may substitute for food reward to limit food intake and suppress weight gain. PMID:25668514

  3. Protease inhibitor-induced nausea and vomiting is attenuated by a peripherally acting, opioid-receptor antagonist in a rat model

    PubMed Central

    Yuan, Chun-Su; Wang, Chong-Zhi; Mehendale, Sangeeta R; Aung, Han H; Foo, Adela; Israel, Robert J

    2009-01-01

    Background Protease inhibitors such as ritonavir can cause nausea and vomiting which is the most common reason for discontinuation. Rats react to nauseous and emetic stimuli by increasing their oral intake of non-nutritive substances like kaolin, known as pica behavior. In this study, we evaluated the effects of methylnaltrexone, a peripherally acting mu-opioid receptor antagonist that does not affect analgesia, on ritonavir-induced nausea and vomiting in a rat pica model. Results We observed that 24 to 48 hr after administration of oral ritonavir 20 mg/kg, kaolin consumption increased significantly in rats (P < 0.01). This increase was attenuated by pretreatment with an intraperitoneal injection of methylnaltrexone (0.3–3.0 mg/kg) in a dose dependent manner (P < 0.01) and also with naloxone (0.1–0.3 mg/kg) (P < 0.01). The areas under the curve for kaolin intake from time 0 to 120 hr were significantly reduced after administration of the opioid antagonists. Food intake was not significantly affected. Plasma naltrexone levels were measured after methylnaltrexone injection, and no detectable levels were found, indicating that methylnaltrexone was not demethylated in our experimental paradigm. Conclusion These results suggest that methylnaltrexone may have potential clinical utility in reducing nausea and vomiting in HIV patients who take ritonavir. PMID:19698111

  4. Thienorphine is a potent long-acting partial opioid agonist: a comparative study with buprenorphine.

    PubMed

    Yu, Gang; Yue, Yong-Juan; Cui, Meng-Xun; Gong, Ze-Hui

    2006-07-01

    A strategy in the development of new treatment for opioid addiction is to find partial opioid agonists with properties of long duration of action and high oral bioavailability. In a search for such compounds, thienorphine, a novel analog of buprenorphine, was synthesized. Here, we reported that, like buprenorphine, thienorphine bound potently and nonselectively to mu-, delta-, and kappa-opioid receptors stably expressed in CHO (Chinese hamster ovary) cells and behaved as a partial agonist at mu-opioid receptor. However, some differences were observed between the pharmacological profiles of thienorphine and buprenorphine. In vitro, thienorphine was more potent than buprenorphine in inhibiting [3H]diprenorphine and stimulating guanosine 5'-O-(3-[35S]thio)triphosphate binding to rat mu-opioid receptor stably expressed in CHO cells. In vivo, thienorphine exhibited a less potent but more efficacious antinociceptive effect with an ED50 value of 0.25 mg/kg s.c. and more potent antimorphine effect with an ED50 value of 0.64 mg/kg intragastric, compared with buprenorphine. Additionally, the bioavailability of thienorphine was greatly higher than that of buprenorphine after oral administration. Moreover, compared with buprenorphine, thienorphine showed a similar long-lasting antinociceptive effect but a much longer antagonism of morphine-induced lethality (more than 15 days). These results indicate that thienorphine is a potent, long-acting partial opioid agonist with high oral bioavailability and may have possible application in treating addiction. PMID:16569757

  5. Antitussive activity of Withania somnifera and opioid receptors.

    PubMed

    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fraňová, Soňa; Ondrejka, Igor; Flešková, Dana

    2015-01-01

    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways. PMID:25252908

  6. Pyrrolo- and Pyridomorphinans: Non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists

    PubMed Central

    Kumar, V.; Clark, M.J.; Traynor, J.R.; Lewis, J.W.; Husbands, S.M.

    2014-01-01

    Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse. PMID:24973818

  7. Pyrrolo- and pyridomorphinans: non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists.

    PubMed

    Kumar, V; Clark, M J; Traynor, J R; Lewis, J W; Husbands, S M

    2014-08-01

    Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse. PMID:24973818

  8. Alterations in the expression of G-proteins and regulation of adenylate cyclase in human neuroblastoma SH-SY5Y cells chronically exposed to low-efficacy mu-opioids.

    PubMed

    Ammer, H; Schulz, R

    1993-10-01

    with low-nanomolar concentrations of guanosine 5'-[beta gamma- imido]triphosphate. Our data demonstrate that chronic treatment of SH-SY5Y cells with low-efficacy mu-opioids increases G-protein abundance, a phenomenon which might contribute to the biochemical mechanisms underlying opioid tolerance/dependence. PMID:8216227

  9. Opioid Receptors Mediate Direct Predictive Fear Learning: Evidence from One-Trial Blocking

    ERIC Educational Resources Information Center

    Cole, Sindy; McNally, Gavan P.

    2007-01-01

    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including [mu]-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear…

  10. Determining Pharmacological Selectivity of the Kappa Opioid Receptor Antagonist LY2456302 Using Pupillometry as a Translational Biomarker in Rat and Human

    PubMed Central

    Witcher, Jennifer W.; Lowe, Stephen L.; Gonzales, Celedon R.; Weller, Mary Ann; Bell, Robert L.; Hart, John C.; Need, Anne B.; McKinzie, Jamie H.; Statnick, Michael A.; Suico, Jeffrey G.; McKinzie, David L.; Tauscher-Wisniewski, Sitra; Mitch, Charles H.; Stoltz, Randall R.; Wong, Conrad J.

    2015-01-01

    Background: Selective kappa opioid receptor antagonism is a promising experimental strategy for the treatment of depression. The kappa opioid receptor antagonist, LY2456302, exhibits ~30-fold higher affinity for kappa opioid receptors over mu opioid receptors, which is the next closest identified pharmacology. Methods: Here, we determined kappa opioid receptor pharmacological selectivity of LY2456302 by assessing mu opioid receptor antagonism using translational pupillometry in rats and humans. Results: In rats, morphine-induced mydriasis was completely blocked by the nonselective opioid receptor antagonist naloxone (3mg/kg, which produced 90% mu opioid receptor occupancy), while 100 and 300mg/kg LY2456302 (which produced 56% and 87% mu opioid receptor occupancy, respectively) only partially blocked morphine-induced mydriasis. In humans, fentanyl-induced miosis was completely blocked by 50mg naltrexone, and LY2456302 dose-dependently blocked miosis at 25 and 60mg (minimal-to-no blockade at 4–10mg). Conclusions: We demonstrate, for the first time, the use of translational pupillometry in the context of receptor occupancy to identify a clinical dose of LY2456302 achieving maximal kappa opioid receptor occupancy without evidence of significant mu receptor antagonism. PMID:25637376

  11. Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming.

    PubMed

    Araldi, Dioneia; Ferrari, Luiz F; Levine, Jon D

    2016-08-01

    We have recently described a novel form of hyperalgesic priming (type II) induced by agonists at two clinically important Gi-protein-coupled receptors (Gi-GPCRs), mu-opioid and A1-adenosine. Like mu-opioids, the antimigraine triptans, which act at 5-HT1B/D Gi-GPCRs, have been implicated in pain chronification. We determined whether sumatriptan, a prototypical 5-HT1B/D agonist, produces type II priming. Characteristic of hyperalgesic priming, intradermal injection of sumatriptan (10 ng) induced a change in nociceptor function such that a subsequent injection of prostaglandin-E2 (PGE2) induces prolonged mechanical hyperalgesia. However, onset to priming was delayed 3 days, characteristic of type I priming. Also characteristic of type I priming, a protein kinase Cε, but not a protein kinase A inhibitor attenuated the prolongation phase of PGE2 hyperalgesia. The prolongation of PGE2 hyperalgesia was also permanently reversed by intradermal injection of cordycepin, a protein translation inhibitor. Also, hyperalgesic priming did not occur in animals pretreated with pertussis toxin or isolectin B4-positive nociceptor toxin, IB4-saporin. Finally, as observed for other agonists that induce type I priming, sumatriptan did not induce priming in female rats. The prolongation of PGE2 hyperalgesia induced by sumatriptan was partially prevented by coinjection of antagonists for the 5-HT1B and 5-HT1D, but not 5-HT7, serotonin receptors and completely prevented by coadministration of a combination of the 5-HT1B and 5-HT1D antagonists. Moreover, the injection of selective agonists, for 5-HT1B and 5-HT1D receptors, also induced hyperalgesic priming. Our results suggest that sumatriptan, which signals through Gi-GPCRs, induces type I hyperalgesic priming, unlike agonists at other Gi-GPCRs, which induce type II priming. PMID:27075428

  12. Different subtypes of opioid receptors have different affinities for G-proteins.

    PubMed

    Polastron, J; Jauzac, P

    1994-05-01

    In this work, we have characterized the opioid receptor expressed by the human neuroblastoma cell line SK-N-BE and compared its hydrodynamic behaviour with those of well known opioid receptors: mu-opioid receptor of rabbit cerebellum and delta-opioid receptor of the hybrid cell line NG 108-15. Human neuroblastoma cell line SK-N-BE expresses a substantial amount of opioid receptors (200-300 fmoles/mg of protein). Pharmacological characterization suggests an heterogenous population of receptors and the presence of two delta subtypes which are, at least partially, negatively coupled with adenylate cyclase via a Gi protein. These receptors exist under two different molecular forms and, in this respect, strikingly contrast with the archetypic delta receptors of NG 108-15 hybrid cell line which show only a high molecular weight form and appear more tightly coupled with the G protein. Hydrodynamic behaviour of SK-N-BE opioid receptors is reminiscent of the profile observed with the rabbit cerebellum mu-opioid receptor. This observation is consistent with the presence of two delta-opioid receptors subtypes, one of which exhibiting properties close to those of mu opioid receptors. Taken overall, our results suggest that different types and subtypes of opioid receptors, even if they are coupled to the same inhibitory G protein, are more or less tightly coupled with their transduction proteins and that closely related opioid receptors can form allosterically interacting complexes. PMID:7920183

  13. Synthetic and Receptor Signaling Explorations of the Mitragyna Alkaloids: Mitragynine as an Atypical Molecular Framework for Opioid Receptor Modulators.

    PubMed

    Kruegel, Andrew C; Gassaway, Madalee M; Kapoor, Abhijeet; Váradi, András; Majumdar, Susruta; Filizola, Marta; Javitch, Jonathan A; Sames, Dalibor

    2016-06-01

    Mu-opioid receptor agonists represent mainstays of pain management. However, the therapeutic use of these agents is associated with serious side effects, including potentially lethal respiratory depression. Accordingly, there is a longstanding interest in the development of new opioid analgesics with improved therapeutic profiles. The alkaloids of the Southeast Asian plant Mitragyna speciosa, represented by the prototypical member mitragynine, are an unusual class of opioid receptor modulators with distinct pharmacological properties. Here we describe the first receptor-level functional characterization of mitragynine and related natural alkaloids at the human mu-, kappa-, and delta-opioid receptors. These results show that mitragynine and the oxidized analogue 7-hydroxymitragynine, are partial agonists of the human mu-opioid receptor and competitive antagonists at the kappa- and delta-opioid receptors. We also show that mitragynine and 7-hydroxymitragynine are G-protein-biased agonists of the mu-opioid receptor, which do not recruit β-arrestin following receptor activation. Therefore, the Mitragyna alkaloid scaffold represents a novel framework for the development of functionally biased opioid modulators, which may exhibit improved therapeutic profiles. Also presented is an enantioselective total synthesis of both (-)-mitragynine and its unnatural enantiomer, (+)-mitragynine, employing a proline-catalyzed Mannich-Michael reaction sequence as the key transformation. Pharmacological evaluation of (+)-mitragynine revealed its much weaker opioid activity. Likewise, the intermediates and chemical transformations developed in the total synthesis allowed the elucidation of previously unexplored structure-activity relationships (SAR) within the Mitragyna scaffold. Molecular docking studies, in combination with the observed chemical SAR, suggest that Mitragyna alkaloids adopt a binding pose at the mu-opioid receptor that is distinct from that of classical opioids. PMID

  14. Receptor reserve reflects differential intrinsic efficacy associated with opioid diastereomers.

    PubMed

    Carliss, Richard D S; Keefer, James F; Perschke, Scott; Welch, Sandra; Rich, Thomas C; Weissman, Arthur D

    2009-05-01

    Structure-activity relationships built around receptor binding or cell-based assays are designed to reveal physiochemical differences between ligands. We hypothesized that agonist receptor reserve may provide a unique approach to distinguish structurally-related agonists exhibiting similar functional characteristics. An intracellular calcium activation assay in Chinese Hamster Ovary (CHO) cells expressing cloned human mu-opioid receptors was developed. We examined two isomers exhibiting indistinguishable receptor binding and in vitro potency profiles. Oxymorphone, a clinically-available congener of codeine has at least two active diastereomeric metabolites (6alpha- and 6beta-oxymorphols) found to be similar for mu-opioid receptor binding affinity (K(d) = 15 versus 14 nM) and calcium activation (EC(50) = 22 versus 14 nM). Calcium activation was then inhibited in CHO cells in a concentration-dependent manner using the irreversible mu-opioid receptor antagonist, beta-funaltrexamine (beta-FNA). Under these conditions, approximately 10-fold greater receptor reserve was found for 6alpha-oxymorphol compared to 6beta-oxymorphol. This difference between the oxymorphols corresponded to a rank order of intrinsic efficacy (Emax): DAMGO > oxymorphone = 6alpha-oxymorphol = oxycodone > 6beta-oxymorphol. In addition, 6alpha-oxymorphol exhibited greater relative potency than the 6beta-oxymorphol in mouse tail-flick, hot-plate and phenylquinone writhing antinociceptive assays, regardless of route of administration. Thus the beta-FNA/calcium model provides a novel, cell-based approach to distinguish structurally related mu-opioid agonists, and in the specific case of the oxymorphols, receptor reserve differences provided a means to bridge functional in vitro and in vivo models. PMID:19463265

  15. Autoradiographic localization of mu and delta opioid receptors in the mesocorticolimbic dopamine system

    SciTech Connect

    Dilts, R.P. Jr.

    1989-01-01

    In vitro autoradiographic techniques were coupled with selective chemical lesions of the A10 dopamine cells and intrinsic perikarya of the region to delineate the anatomical localization of mu and delta opioid receptors, as well as, neurotensin receptors. Mu opioid receptors were labeled with {sup 125}I-DAGO. Delta receptors were labeled with {sup 125}I-DPDPE. Neurotensin receptors were labeled with {sup 125}I-NT3. Unilateral lesions of the dopamine perikarya were produced by injections of 6-OHDA administered in the ventral mesencephalon. Unilateral lesions of intrinsic perikarya were induced by injections of quinolinic acid in to the A10 dopamine cell region. Unilateral lesions produced with 6-OHDA resulted in the loss of neurotensin receptors in the A10 region and within the terminal fields. Mu opioid receptors were unaffected by this treatment, but delta opioid receptors increased in the contralateral striatum and nucleus accumbens following 6-OHDA administration. Quinolinic acid produced a reduction of mu opioid receptors within the A10 region with a concomitant reduction in neurotensin receptors in both the cell body region and terminal fields. These results are consistent with a variety of biochemical and behavioral data which suggest the indirect modulation of dopamine transmission by the opioids. In contrast these results strongly indicate a direct modulation of the mesolimbic dopamine system by neurotensin.

  16. Sigma 1 receptor modulation of G-protein-coupled receptor signaling: potentiation of opioid transduction independent from receptor binding.

    PubMed

    Kim, Felix J; Kovalyshyn, Ivanka; Burgman, Maxim; Neilan, Claire; Chien, Chih-Cheng; Pasternak, Gavril W

    2010-04-01

    sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling. PMID:20089882

  17. Comparison of [Dmt1]DALDA and DAMGO in binding and G protein activation at mu, delta, and kappa opioid receptors.

    PubMed

    Zhao, Guo-Min; Qian, Xuanxuan; Schiller, Peter W; Szeto, Hazel H

    2003-12-01

    [Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine) binds with high affinity and selectivity to the mu opioid receptor and is a surprisingly potent and long-acting analgesic, especially after intrathecal administration. In an attempt to better understand the unique pharmacological profile of [Dmt1]DALDA, we have prepared [3H][Dmt1]DALDA and compared its binding properties with that of [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Kinetic studies revealed rapid association of [3H][Dmt1]DALDA when incubated with mouse brain membranes (K+1 = 0.155 nM(-1) min(-1)). Dissociation of [3H][Dmt1]DALDA was also rapid (K(-1) = 0.032 min(-1)) and indicated binding to a single site. [3H][Dmt1]DALDA binds with very high affinity to human mu opioid receptor (hMOR) (Kd = 0.199 nM), and Kd and Bmax were reduced by sodium but not Gpp(NH)p [guanosine 5'-(beta,gamma-imido)triphosphate]. Similar Kd values were obtained in brain and spinal cord tissues and SH-SY5Y cells. The hMOR:hDOR (human delta opioid receptor) selectivity of [Dmt1]DALDA ( approximately 10,000) is 8-fold higher than DAMGO. However, [Dmt1]DALDA is less selective than DAMGO against hKOR (human kappa opioid receptor) (26-versus 180-fold). The Ki values for a number of opioid ligands were generally higher when determined by competitive displacement binding against [3H][Dmt1]DALDA compared with [3H]DAMGO, with the exception of Dmt1-substituted peptide analogs. All Dmt1 analogs showed much higher affinity for the mu receptor than corresponding Tyr1 analogs. [35S]GTPgammaS (guanosine 5'-O -(3-[35S]thio)triphosphate) binding showed that [Dmt1]DALDA and DAMGO are full agonists at hMOR and hDOR but are only partial agonists at hKOR. The very high affinity and selectivity of [3H][Dmt1]DALDA for the mu receptor, together with its very low nonspecific binding (10-15%) and metabolic stability, make [3H][Dmt1]DALDA an ideal radioligand for labeling mu receptors. PMID:14534366

  18. Ionic basis of cold receptors acting as thermostats.

    PubMed

    Okazawa, Makoto; Takao, Keizo; Hori, Aiko; Shiraki, Takuma; Matsumura, Kiyoshi; Kobayashi, Shigeo

    2002-05-15

    When temperature (T) of skin decreases stepwise, cold fibers evoke transient afferent discharges, inducing cold sensation and heat-gain responses. Hence we have proposed that cold receptors at distal ends of cold fibers are thermostats to regulate skin T against cold. Here, with patch-clamp techniques, we studied the ionic basis of cold receptors in cultured dorsal root ganglion (DRG) neurons of rats, as a model of nerve endings. Cells that increased cytosolic Ca(2+) level in response to moderate cooling were identified as neurons with cold receptors. In whole-cell current-clamp recordings of these cells, in response to cooling, cold receptors evoked a dynamic receptor potential (RP), eliciting impulses briefly. In voltage-clamp recordings (-60 mV), step cooling induced an inward cold current (I(cold)) with inactivation, underlying the dynamic RP. Ca(2+) ions that entered into cells from extracellular side induced the inactivation. Analysis of the reversal potential implied that I(cold) was nonselective cation current with high Ca(2+) permeability. Threshold temperatures of cooling-induced Ca(2+) response and I(cold) were different primarily among cells. In outside-out patches, when T decreased, single nonselective cation channels became active at a critical T. This implies that a cold receptor is an ion channel and acts as the smallest thermostat. Because these thermal properties were consistent with that in cold fibers, we conclude that the same cold receptors exist at nerve endings and generate afferent impulses for cold sensation and heat-gain behaviors in response to cold. PMID:12019319

  19. Methylnaltrexone Injection

    MedlinePlus

    ... injection is used to treat constipation caused by opioid (narcotic) pain medications in patients with advanced illnesses ... a class of medications called peripherally acting mu-opioid receptor antagonists. It works by protecting the bowel ...

  20. Alvimopan

    MedlinePlus

    ... a class of medications called peripherally acting mu-opioid receptor antagonists. It works by protecting the bowel from the constipation effects of opioid (narcotic) medications that are used to treat pain ...

  1. Design, Syntheses, and Pharmacological Characterization of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan Analogues as Opioid Receptor Ligands

    PubMed Central

    Yuan, Yunyun; Zaidi, Saheem A.; Stevens, David L.; Scoggins, Krista L.; Mosier, Philip D.; Kellogg, Glen E.; Dewey, William L.; Selley, Dana E.; Zhang, Yan

    2015-01-01

    A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1′- and/or 4′-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In contrast, substitutions at the 6′-and/or 7′-position of the isoquinoline ring reduced MOR selectivity as well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca2+ increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indicated orientation of the 6’-nitro group varied significantly in the different “address” domains of the receptors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current findings provide valuable insights for future development of NAQ-based MOR selective ligands. PMID:25783191

  2. Design, syntheses, and pharmacological characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan analogues as opioid receptor ligands.

    PubMed

    Yuan, Yunyun; Zaidi, Saheem A; Stevens, David L; Scoggins, Krista L; Mosier, Philip D; Kellogg, Glen E; Dewey, William L; Selley, Dana E; Zhang, Yan

    2015-04-15

    A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1'- and/or 4'-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In contrast, substitutions at the 6'- and/or 7'-position of the isoquinoline ring reduced MOR selectivity as well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca(2+) increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indicated orientation of the 6'-nitro group varied significantly in the different 'address' domains of the receptors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current findings provide valuable insights for future development of NAQ-based MOR selective ligands. PMID:25783191

  3. Activity profiles of dalargin and its analogues in mu-, delta- and kappa-opioid receptor selective bioassays.

    PubMed

    Pencheva, N; Pospisek, J; Hauzerova, L; Barth, T; Milanov, P

    1999-10-01

    1. To elucidate the structural features ensuring action of [D-Ala2, Leu5]-enkephalyl-Arg (dalargin), a series of dalargin analogues were tested for their effectiveness in depressing electrically-evoked contractions of the guinea-pig myenteric plexus-longitudinal muscle preparations (mu- and kappa-opioid receptors) and the vasa deferentia of the hamster (delta-opioid receptors), mouse (mu-, delta- and kappa-opioid receptors), rat (similar to mu-opioid receptors) and rabbit (kappa-opioid receptors). The naloxone KB values in the myenteric plexus were also obtained. 2. [L-Ala2]-dalargin was 19 times less potent than dalargin, and its pharmacological activity was peptidase-sensitive. The ratio of delta-activity to mu-activity for [L-Ala2]-dalargin was 6.78, and KB was 7.9 nM. This emphasizes the role that D-configuration of Ala2 plays in determining the active folding of dalargin molecule as well as in conferring resistance to peptidases. 3. [Met5]-dalargin was equipotent to dalargin in the myenteric plexus, but was more potent in the vasa deferentia of hamster and mouse (KB=5.5 nM). Leu5 and the interdependence of Leu5 and D-Ala2 are of importance for the selectivity of dalargin for mu-opioid receptors. 4. Dalarginamide was more potent and selective for mu-opioid receptors than dalargin, whilst dalarginethylamide, though equipotent to dalarginamide in the myenteric plexus, was more potent at delta-opioid receptors (KB=5.0 nM). [D-Phe4]-dalarginamide and N-Me-[D-Phe4]-dalarginamide were inactive indicating the contribution of L-configuration of Phe4 to the pharmacological potency of dalargin. 5. N-Me-[L-Phe4]-dalarginamide possessed the highest potency and selectivity for mu-opioid receptors (the ratio of delta-activity to mu-activity was 0.00053; KB=2.6 nM). The CONH2 terminus combined with the N-methylation of L-Phe4 increased the potency and selectivity of dalargin for mu-opioid receptors. PMID:10516634

  4. Cryptochinones from Cryptocarya chinensis act as farnesoid X receptor agonists.

    PubMed

    Lin, Hsiang-Ru; Chou, Tsung-Hsien; Huang, Din-Wen; Chen, Ih-Sheng

    2014-09-01

    Cryptochinones A-D are tetrahydroflavanones isolated from the leaves of Cryptocarya chinensis, an evergreen tree whose extracts are believed to have a variety of health benefits. The origin of their possible bioactivity is unclear. The farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that has been widely targeted for developing treatments for chronic liver disease and for hyperglycemia. We studied whether cryptochinones A-D, which are structurally similar to known FXR ligands, may act at this target. Indeed, in mammalian one-hybrid and transient transfection reporter assays, cryptochinones A-D transactivated FXR to modulate promoter action including GAL4, SHP, CYP7A1, and PLTP promoters in dose-dependent manner, while they exhibited similar agonistic activity as chenodeoxycholic acid (CDCA), an endogenous FXR agonist. Through molecular modeling docking studies we evaluated their ability to bind to the FXR ligand binding pocket. Our results indicate that cryptochinones A-D can behave as FXR agonists. PMID:25127166

  5. APJ acts as a dual receptor in cardiac hypertrophy.

    PubMed

    Scimia, Maria Cecilia; Hurtado, Cecilia; Ray, Saugata; Metzler, Scott; Wei, Ke; Wang, Jianming; Woods, Chris E; Purcell, Nicole H; Catalucci, Daniele; Akasaka, Takeshi; Bueno, Orlando F; Vlasuk, George P; Kaliman, Perla; Bodmer, Rolf; Smith, Layton H; Ashley, Euan; Mercola, Mark; Brown, Joan Heller; Ruiz-Lozano, Pilar

    2012-08-16

    Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy. PMID:22810587

  6. APJ ACTS AS A DUAL RECEPTOR IN CARDIAC HYPERTROPHY

    PubMed Central

    Scimia, Maria Cecilia; Hurtado, Cecilia; Ray, Saugata; Metzler, Scott; Wei, Ke; Wang, Jianming; Woods, Chris E.; Purcell, Nicole H.; Catalucci, Daniele; Akasaka, Takashi; Bueno, Orlando F.; Vlasuk, George P.; Kaliman, Perla; Bodmer, Rolf; Smith, Layton H.; Ashley, Euan; Mercola, Mark; Brown, Joan Heller; Ruiz-Lozano, Pilar

    2012-01-01

    Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues1. Here we report that genetic loss of APJ confers resistance to chronic pressure overload by dramatically reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechano-sensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and for the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy. PMID:22810587

  7. Metalloproteolytic receptor shedding…platelets "acting their age".

    PubMed

    Andrews, Robert K; Gardiner, Elizabeth E

    2016-09-01

    Whilst significant effort has been focused on development of tools and approaches to clinically modulate activation processes that consume platelets, the platelet receptors that initiate activation processes remain untargeted. The modulation of receptor levels is also linked to underlying platelet aging processes which influence normal platelet lifespan and also the functionality and survival of stored platelets that are used in transfusion. In this review, we will focus on platelet adhesion receptors initiating thrombus formation, and discuss how regulation of levels of these receptors impact platelet function and platelet survival. PMID:27459696

  8. Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors

    SciTech Connect

    Jin,R.; Clark, S.; Weeks, A.; Dudman, J.; Gouaux, E.; Partin, K.

    2005-01-01

    Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1, 3-oxazino benzo-1, 4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the 'hinge' in the ligand-binding core 'clamshell' that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

  9. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice.

    PubMed

    Vang, Derek; Paul, Jinny A; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T; Gupta, Kalpna

    2015-12-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

  10. Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice

    PubMed Central

    Vang, Derek; Paul, Jinny A.; Nguyen, Julia; Tran, Huy; Vincent, Lucile; Yasuda, Dennis; Zaveri, Nurulain T.; Gupta, Kalpna

    2015-01-01

    Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

  11. Role of Mu and Delta Opioid Receptors in the Nucleus Accumbens in Cocaine-Seeking Behavior

    PubMed Central

    Simmons, Diana; Self, David W.

    2009-01-01

    Previous studies suggest that opioid receptors in the ventral tegmental area (VTA), but not the nucleus accumbens (NAc), play a role in relapse to drug-seeking behavior. However, environmental stimuli that elicit relapse also release the endogenous opioid β-endorphin in the NAc. Using a within–session extinction/reinstatement paradigm in rats that self-administer cocaine, we found that NAc infusions of the mu opioid receptor (MOR) agonist DAMGO moderately reinstated responding on the cocaine-paired lever at low doses (1.0–3.0 ng/side), whereas the delta opioid receptor (DOR) agonist DPDPE induced greater responding at higher doses (300–3000 ng/side) that also enhanced inactive lever responding. Using doses of either agonist that induced responding on only the cocaine-paired lever, we found that DAMGO-induced responding was blocked selectively by pretreatment with the MOR antagonist CTAP, while DPDPE-induced responding was selectively blocked by the DOR antagonist naltrindole. Cocaine-primed reinstatement was blocked by intra-NAc CTAP but not naltrindole, indicating a role for endogenous MOR-acting peptides in cocaine-induced reinstatement of cocaine-seeking behavior. In this regard, intra-NAc infusions of β-endorphin (100–1000 ng/side) induced marked cocaine-seeking behavior, an effect blocked by intra-NAc pretreatment with the MOR but not DOR antagonist. Conversely, cocaine seeking elicited by the enkephalinase inhibitor thiorphan (1–10 μg/side) was blocked by naltrindole but not CTAP. MOR stimulation in more dorsal caudate-putamen sites was ineffective, while DPDPE infusions induced cocaine seeking. Together, these findings establish distinct roles for MOR and DOR in cocaine relapse, and suggest that NAc MOR could be an important therapeutic target to neutralize the effects of endogenous β-endorphin release on cocaine relapse. PMID:19279569

  12. Ligands for opioid and sigma-receptors improve cardiac electrical stability in rat models of post-infarction cardiosclerosis and stress.

    PubMed

    Lishmanov YuB; Maslov, L N; Naryzhnaya, N V; Tam, S W

    1999-01-01

    The effects of the extremely selective mu-opioid receptor agonist, [D-Arg2,Lys4]-dermorphin-(1-4)-amide (DALDA), the mu-opioid receptor agonist morphine, the mu/delta agonist D-Ala2, Leu5, Arg6-enkephalin (dalargin), the kappa-opioid receptor agonist spiradoline, and the sigma1-receptor antagonist DuP 734 on ventricular fibrillation threshold (VFT) was investigated in an experimental post-infarction cardiosclerosis model and an immobilization stress-induced model in rats. Both models produced a significant decrease in VFT. The postinfarction cardiosclerosis-induced decrease in VFT was significantly reversed by intravenous administration of dalargin (0.1 mg/kg), DALDA (0.1 mg/kg), or morphine HCl (1.5 mg/kg). Pretreatment with naloxone (0.2 mg/kg) completely eliminated the increase in cardiac electrical stability produced by DALDA. Both spiradoline (8 mg/kg, i.p.) and DuP 734 (1 mg/kg, i.p.) produced a significant increase in VFT in rats with post-infarction cardiosclerosis. This effect of spiradoline was blocked by nor-binaltorphimine. The immobilization stress-induced decrease in VFT was significantly reversed by administration of either DALDA, spiradoline or DuP 734. In conclusion, activation of either mu- or kappa1-opioid receptors or blockade of sigma1-receptors reversed the decrease in VFT in both cardiac compromised models. Since DALDA and dalargin essentially do not cross blood brain barriers, their effects on VFT may be mediated through peripheral mu-opioid receptors. PMID:10403501

  13. Regulation of opioid receptors by cocaine.

    PubMed

    Unterwald, E M

    2001-06-01

    Cocaine is a widely abused psychostimulant. Its direct actions include inhibition of dopamine, serotonin, and norepinephrine reuptake into presynaptic nerve terminals, thereby potentiating the actions of these transmitters in the synapse. A variety of studies have demonstrated that cocaine can also have profound effects on the endogenous opioid system. Compelling evidence points to the importance of mu opioid receptors in human cocaine addiction and craving. Animal studies support these findings and demonstrate that chronic cocaine administration can result in alterations in opioid receptor expression and function as measured by changes in critical signal transduction pathways. This chapter reviews studies on the regulation of opioid receptors as the result of exposure to cocaine. PMID:11458541

  14. Morphine-induced desensitization and down-regulation at mu-receptors in 7315C pituitary tumor cells

    SciTech Connect

    Puttfarcken, P.S.; Cox, B.M. )

    1989-01-01

    Pituitary 7315c tumor cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 {mu}M, for periods of five or forty-eight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed membrane preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same membrane preparations, the number and affinity of mu-opioid receptors was estimated by measurements of ({sup 3}H)diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for ({sup 3}H)diprenorphine. However after 48 hr of morphine treatment, greater than 25% reductions in receptor number were apparent with morphine pretreatment concentrations of 10 {mu}M or higher. These results suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor number occurred after exposure to high concentrations of morphine for periods longer than 5hr.

  15. Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems.

    PubMed

    Toll, Lawrence; Bruchas, Michael R; Calo', Girolamo; Cox, Brian M; Zaveri, Nurulain T

    2016-04-01

    The NOP receptor (nociceptin/orphanin FQ opioid peptide receptor) is the most recently discovered member of the opioid receptor family and, together with its endogenous ligand, N/OFQ, make up the fourth members of the opioid receptor and opioid peptide family. Because of its more recent discovery, an understanding of the cellular and behavioral actions induced by NOP receptor activation are less well developed than for the other members of the opioid receptor family. All of these factors are important because NOP receptor activation has a clear modulatory role on mu opioid receptor-mediated actions and thereby affects opioid analgesia, tolerance development, and reward. In addition to opioid modulatory actions, NOP receptor activation has important effects on motor function and other physiologic processes. This review discusses how NOP pharmacology intersects, contrasts, and interacts with the mu opioid receptor in terms of tertiary structure and mechanism of receptor activation; location of receptors in the central nervous system; mechanisms of desensitization and downregulation; cellular actions; intracellular signal transduction pathways; and behavioral actions with respect to analgesia, tolerance, dependence, and reward. This is followed by a discussion of the agonists and antagonists that have most contributed to our current knowledge. Because NOP receptors are highly expressed in brain and spinal cord and NOP receptor activation sometimes synergizes with mu receptor-mediated actions and sometimes opposes them, an understanding of NOP receptor pharmacology in the context of these interactions with the opioid receptors will be crucial to the development of novel therapeutics that engage the NOP receptor. PMID:26956246

  16. The multiple facets of opioid receptor function: implications for addiction

    PubMed Central

    Lutz, Pierre-Eric; Kieffer, Brigitte L.

    2013-01-01

    Addiction is characterized by altered reward processing, disrupted emotional responses and poor decision-making. Beyond a central role in drug reward, increasing evidence indicate that opioid receptors are more generally involved in all these processes. Recent studies establish the mu opioid receptor as a main player in social reward, which attracts increasing attention in psychiatric research. There is growing interest in blocking the kappa opioid receptor to prevent relapse, and alleviate the negative affect of withdrawal. The delta opioid receptor emerges as a potent mood enhancer, whose involvement in addiction is less clear. All three opioid receptors are likely implicated in addiction-depression comorbidity, and understanding of their roles in cognitive deficits associated to drug abuse is only beginning. PMID:23453713

  17. Differences in acute anorectic effects of long-acting GLP-1 receptor agonists in rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists have both glucose- and weight-lowering effects. The brain is poised to mediate both of these actions since GLP-1Rs are present in key areas known to control weight and glucose. Although some research has been performed on the effects of ...

  18. Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells.

    PubMed

    Barrios-García, Tonatiuh; Gómez-Romero, Vania; Tecalco-Cruz, Ángeles; Valadéz-Graham, Viviana; León-Del-Río, Alfonso

    2016-06-01

    Tristetraprolin (TTP) is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα), which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR), glucocorticoid receptor (GR) and androgen receptor (AR). In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors. PMID:27114912

  19. Nuclear tristetraprolin acts as a corepressor of multiple steroid nuclear receptors in breast cancer cells

    PubMed Central

    Barrios-García, Tonatiuh; Gómez-Romero, Vania; Tecalco-Cruz, Ángeles; Valadéz-Graham, Viviana; León-Del-Río, Alfonso

    2016-01-01

    Tristetraprolin (TTP) is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα), which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR), glucocorticoid receptor (GR) and androgen receptor (AR). In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors. PMID:27114912

  20. Activity of new NOP receptor ligands in a rat peripheral mononeuropathy model: Potentiation of Morphine anti-allodynic activity by NOP receptor antagonists

    PubMed Central

    Khroyan, Taline V.; Polgar, Willma E.; Orduna, Juan; Jiang, Faming; Olsen, Cris; Toll, Lawrence; Zaveri, Nurulain T.

    2009-01-01

    The effect of new NOP receptor agonists and antagonists in the rat chronic constriction injury model was investigated. Intraperitoneally administered NOP receptor agonist SR14150 and antagonists SR16430 and SR14148, had no effect on mechanical allodynia when given alone. The nonselective NOP/mu-opioid receptor agonist SR16435, however, produced an anti-allodynic response, similar to morphine and reversible by naloxone. Notably, co-administration of the NOP receptor antagonists potentiated the anti-allodynic activity of both morphine and SR16435. Increased levels of the NOP receptor are implicated in the reduced efficacy of morphine in neuropathic pain. Our results suggest the utility of NOP receptor antagonists for potentiating opioid efficacy in chronic pain. PMID:19285491

  1. The liver X receptor agonist T0901317 acts as androgen receptor antagonist in human prostate cancer cells

    SciTech Connect

    Chuu, Chih-pin; Chen, Rou-Yu; Hiipakka, Richard A.; Kokontis, John M.; Warner, Karen V.; Xiang, Jialing; Liao, Shutsung . E-mail: sliao@uchicago.edu

    2007-06-01

    T0901317 is a potent non-steroidal synthetic liver X receptor (LXR) agonist. T0901317 blocked androgenic stimulation of the proliferation of androgen-dependent LNCaP 104-S cells and androgenic suppression of the proliferation of androgen-independent LNCaP 104-R2 cells, inhibited the transcriptional activation of an androgen-dependent reporter gene by androgen, and suppressed gene and protein expression of prostate specific antigen (PSA), a target gene of androgen receptor (AR) without affecting gene and protein expression of AR. T0901317 also inhibited binding of a radiolabeled androgen to AR, but inhibition was much weaker compared to the effect of the antiandrogens, bicalutamide and hydroxyflutamide. The LXR agonist T0901317, therefore, acts as an antiandrogen in human prostate cancer cells.

  2. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    PubMed

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms. PMID:25330347

  3. Lepidozenolide from the liverwort Lepidozia fauriana acts as a farnesoid X receptor agonist.

    PubMed

    Lin, Hsiang-Ru

    2015-01-01

    Lepidozenolide is a sesquiterpenoid isolated from the liverwort Lepidozia fauriana and its possible bioactivity is unclear. The farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that has been widely targeted for developing treatments for chronic liver disease and hyperglycemia. In this study, whether lepidozenolide may act as a FXR agonist was determined. Indeed, in mammalian one-hybrid and transient transfection reporter assays, lepidozenolide transactivated FXR to modulate promoter action including GAL4, CYP7A1, and PLTP promoters in a dose-dependent manner, while it exhibited slightly less agonistic activity than chenodeoxycholic acid, an endogenous FXR agonist. Through the molecular modeling docking studies lepidozenolide was shown to bind to FXR ligand binding pocket fairly well. All these results indicate that lepidozenolide acts as a FXR agonist. PMID:25315435

  4. Identification of short-acting κ-opioid receptor antagonists with anxiolytic-like activity.

    PubMed

    Peters, Matthew F; Zacco, Anna; Gordon, John; Maciag, Carla M; Litwin, Linda C; Thompson, Carolann; Schroeder, Patricia; Sygowski, Linda A; Piser, Timothy M; Brugel, Todd A

    2011-07-01

    The κ-opioid receptor plays a central role in mediating the response to stressful life events. Inhibiting κ-opioid receptor signaling is proposed as a mechanism for treating stress-related conditions such as depression and anxiety. Preclinical testing consistently confirms that disruption of κ-opioid signaling is efficacious in animal models of mood disorders. However, concerns about the feasibility of developing antagonists into drugs stem from an unusual pharmacodynamic property of prototypic κ-opioid receptor-selective antagonists; they inhibit receptor signaling for weeks to months after a single dose. Several fundamental questions include - is it possible to identify short-acting antagonists; is long-lasting inhibition necessary for efficacy; and is it safe to develop long-acting antagonists in the clinic. Here, we test representative compounds (AZ-ECPC, AZ-MTAB, and LY-DMPF) from three new chemical series of κ-opioid receptor ligands for long-lasting inhibition. Each compound dose-dependently reversed κ-opioid agonist-induced diuresis. However, unlike the prototypic antagonist, nBNI, which fully inhibited evoked diuresis for at least four weeks, the new compounds showed no inhibition after one week. The two compounds with greater potency and selectivity were tested in prenatally-stressed rats on the elevated plus maze, an exploration-based model of anxiety. Spontaneous exploration of open arms in the elevated plus maze was suppressed by prenatal stress and restored with both compounds. These findings indicate that persistent inhibition is not an inherent property of κ-opioid-selective antagonists and that post-stress dosing with transient inhibitors can be effective in a mood disorder model. This further supports κ-opioid receptor as a promising target for developing novel psychiatric medications. PMID:21539838

  5. Auxiliary Subunit GSG1L Acts to Suppress Calcium-Permeable AMPA Receptor Function

    PubMed Central

    McGee, Thomas P.; Bats, Cécile

    2015-01-01

    AMPA-type glutamate receptors are ligand-gated cation channels responsible for a majority of the fast excitatory synaptic transmission in the brain. Their behavior and calcium permeability depends critically on their subunit composition and the identity of associated auxiliary proteins. Calcium-permeable AMPA receptors (CP-AMPARs) contribute to various forms of synaptic plasticity, and their dysfunction underlies a number of serious neurological conditions. For CP-AMPARs, the prototypical transmembrane AMPAR regulatory protein stargazin, which acts as an auxiliary subunit, enhances receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines. We find that, in contrast, GSG1L, a transmembrane auxiliary protein identified recently as being part of the AMPAR proteome, acts to reduce the weighted mean single-channel conductance and calcium permeability of recombinant CP-AMPARs, while increasing polyamine-dependent rectification. To examine the effects of GSG1L on native AMPARs, we manipulated its expression in cerebellar and hippocampal neurons. Transfection of GSG1L into mouse cultured cerebellar stellate cells that lack this protein increased the inward rectification of mEPSCs. Conversely, shRNA-mediated knockdown of endogenous GSG1L in rat cultured hippocampal pyramidal neurons led to an increase in mEPSC amplitude and in the underlying weighted mean single-channel conductance, revealing that GSG1L acts to suppress current flow through native CP-AMPARs. Thus, our data suggest that GSG1L extends the functional repertoire of AMPAR auxiliary subunits, which can act not only to enhance but also diminish current flow through their associated AMPARs. SIGNIFICANCE STATEMENT Calcium-permeable AMPA receptors (CP-AMPARs) are an important group of receptors for the neurotransmitter glutamate. These receptors contribute to various forms of

  6. Chicken lutropin acts like follitropin in rat ovarian follitropin receptor: an isoelectric focusing study.

    PubMed

    Iwasawa, A; Hattori, M; Fukuhara, Y; Kawashima, M; Wakabayashi, K; Kamiyoshi, M

    1998-07-01

    This study investigates whether chicken lutropin (LH) specifically binds to rat ovarian follitropin (FSH) receptor and exerts FSH-like bioactivity. Glycoprotein fraction, prepared from the chicken anterior pituitary gland, was fractionated using isoelectric focusing within a pH range of 3.5-11. Analysis of the focused fractions, by a radioreceptor assay (RRA) specific for FSH in rats using rat ovarian homogenate as receptor source, and 125I-labeled rat FSH as radioligand, detected a large component having an isoelectric point of 10.25. This focusing profile obtained by RRA was quite similar to that obtained by a specific radioimmunoassay (RIA) for chicken LH, but clearly different from that obtained by a specific RIA for chicken FSH, indicating this RRA specifically recognizes chicken LH. Chicken LH fraction prepared from the electrofocused material was used for further studies. The chicken LH preparation was three times more potent than rat FSH in the RRA in displacing the radioligand bound to rat ovarian receptor, while chicken LH facilitated an 8-fold less production of estradiol in dispersed rat granulosa cells than rat FSH. These results suggest that chicken LH acts like rat FSH in rat ovarian FSH receptor, but receptor-binding activity is much higher than biological activity. PMID:9827020

  7. Fos-Zippered GH Receptor Cytosolic Tails Act as Jak2 Substrates and Signal Transducers.

    PubMed

    Nespital, Tobias; van der Velden, Lieke M; Mensinga, Anneloes; van der Vaart, Elisabeth D; Strous, Ger J

    2016-03-01

    Members of the Janus kinase (Jak) family initiate the majority of downstream signaling events of the cytokine receptor family. The prevailing principle is that the receptors act in dimers: 2 Jak2 molecules bind to the cytosolic tails of a cytokine receptor family member and initiate Jak-signal transducer and activator of transcription signaling upon a conformational change in the receptor complex, induced by the cognate cytokine. Due to the complexity of signaling complexes, there is a strong need for in vitro model systems. To investigate the molecular details of the Jak2 interaction with the GH receptor (GHR), we used cytosolic tails provided with leucine zippers derived from c-Fos to mimic the dimerized state of GHR. Expressed together with Jak2, fos-zippered tails, but not unzippered tails, were stabilized. In addition, the Jak-signal transducer and activator of transcription signaling pathway was activated by the fos-zippered tails. The stabilization depended also on α-helix rotation of the zippers. Fos-zippered GHR tails and Jak2, both purified from baculovirus-infected insect cells, interacted via box1 with a binding affinity of approximately 40nM. As expected, the Jak kinase inhibitor Ruxolitinib inhibited the stabilization but did not affect the c-Fos-zippered GHR tail-Jak2 interaction. Analysis by blue-native gel electrophoresis revealed high molecular-weight complexes containing both Jak2 and nonphosphorylated GHR tails, whereas Jak2-dissociated tails were highly phosphorylated and monomeric, implying that Jak2 detaches from its substrate upon phosphorylation. PMID:26859362

  8. Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration.

    PubMed

    Harris, Katherine E; Beckendorf, Steven K

    2007-06-01

    Guided cell migration is necessary for the proper function and development of many tissues, one of which is the Drosophila embryonic salivary gland. Here we show that two distinct Wnt signaling pathways regulate salivary gland migration. Early in migration, the salivary gland responds to a WNT4-Frizzled signal for proper positioning within the embryo. Disruption of this signal, through mutations in Wnt4, frizzled or frizzled 2, results in misguided salivary glands that curve ventrally. Furthermore, disruption of downstream components of the canonical Wnt pathway, such as dishevelled or Tcf, also results in ventrally curved salivary glands. Analysis of a second Wnt signal, which acts through the atypical Wnt receptor Derailed, indicates a requirement for Wnt5 signaling late in salivary gland migration. WNT5 is expressed in the central nervous system and acts as a repulsive signal, needed to keep the migrating salivary gland on course. The receptor for WNT5, Derailed, is expressed in the actively migrating tip of the salivary glands. In embryos mutant for derailed or Wnt5, salivary gland migration is disrupted; the tip of the gland migrates abnormally toward the central nervous system. Our results suggest that both the Wnt4-frizzled pathway and a separate Wnt5-derailed pathway are needed for proper salivary gland migration. PMID:17507403

  9. Synergistically acting agonists and antagonists of G protein–coupled receptors prevent photoreceptor cell degeneration

    PubMed Central

    Chen, Yu; Palczewska, Grazyna; Masuho, Ikuo; Gao, Songqi; Jin, Hui; Dong, Zhiqian; Gieser, Linn; Brooks, Matthew J.; Kiser, Philip D.; Kern, Timothy S.; Martemyanov, Kirill A.; Swaroop, Anand; Palczewski, Krzysztof

    2016-01-01

    Photoreceptor cell degeneration leads to visual impairment and blindness in several types of retinal disease. However, the discovery of safe and effective therapeutic strategies conferring photoreceptor cell protection remains challenging. Targeting distinct cellular pathways with low doses of different drugs that produce a functionally synergistic effect could provide a strategy for preventing or treating retinal dystrophies. We took a systems pharmacology approach to identify potential combination therapies using a mouse model of light-induced retinal degeneration. We showed that a combination of U.S. Food and Drug Administration–approved drugs that act on different G protein (guanine nucleotide–binding protein)–coupled receptors (GPCRs) exhibited synergistic activity that protected retinas from light-induced degeneration even when each drug was administered at a low dose. In functional assays, the combined effects of these drugs were stimulation of Gi/o signaling by activating the dopamine receptors D2R and D4R, as well as inhibition of Gs and Gq signaling by antagonizing D1R and the α1A-adrenergic receptor ADRA1A, respectively. Moreover, transcriptome analyses demonstrated that such combined GPCR-targeted treatments preserved patterns of retinal gene expression that were more similar to those of the normal retina than did higher-dose monotherapy. Our study thus supports a systems pharmacology approach to identify treatments for retinopathies, an approach that could extend to other complex disorders. PMID:27460988

  10. Synergistically acting agonists and antagonists of G protein-coupled receptors prevent photoreceptor cell degeneration.

    PubMed

    Chen, Yu; Palczewska, Grazyna; Masuho, Ikuo; Gao, Songqi; Jin, Hui; Dong, Zhiqian; Gieser, Linn; Brooks, Matthew J; Kiser, Philip D; Kern, Timothy S; Martemyanov, Kirill A; Swaroop, Anand; Palczewski, Krzysztof

    2016-01-01

    Photoreceptor cell degeneration leads to visual impairment and blindness in several types of retinal disease. However, the discovery of safe and effective therapeutic strategies conferring photoreceptor cell protection remains challenging. Targeting distinct cellular pathways with low doses of different drugs that produce a functionally synergistic effect could provide a strategy for preventing or treating retinal dystrophies. We took a systems pharmacology approach to identify potential combination therapies using a mouse model of light-induced retinal degeneration. We showed that a combination of U.S. Food and Drug Administration-approved drugs that act on different G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) exhibited synergistic activity that protected retinas from light-induced degeneration even when each drug was administered at a low dose. In functional assays, the combined effects of these drugs were stimulation of Gi/o signaling by activating the dopamine receptors D2R and D4R, as well as inhibition of Gs and Gq signaling by antagonizing D1R and the α1A-adrenergic receptor ADRA1A, respectively. Moreover, transcriptome analyses demonstrated that such combined GPCR-targeted treatments preserved patterns of retinal gene expression that were more similar to those of the normal retina than did higher-dose monotherapy. Our study thus supports a systems pharmacology approach to identify treatments for retinopathies, an approach that could extend to other complex disorders. PMID:27460988

  11. Nucleolin Acts as a Scavenger Receptor for Acetylated Low-Density Lipoprotein on Macrophages.

    PubMed

    Miki, Yuichi; Tachibana, Yoshihiro; Ohminato, Yukari; Fujiwara, Yasuyuki

    2015-01-01

    Although macrophage phagocytoses modified low-density lipoprotein (LDL), excessive accumulation of modified LDL induces macrophage foam cell formation, which is a feature of atherosclerotic plaque. Thus, the identification of scavenger receptor for modified LDL will provide better understanding of an atherosclerotic event. We recently showed that nucleolin expressed on macrophages acts as a scavenger receptor for various endogenous discarded products. Here, we investigated whether or not nucleolin is involved in the uptake of acetylated LDL (AcLDL). In contrast to normal LDL, AcLDL directly bound to immobilized nucleolin. AcLDL exhibited a higher affinity for macrophages than normal LDL. This binding of AcLDL was inhibited by anti-nucleolin antibody and antineoplastic guanine-rich oligonucleotide (AGRO), a nucleolin-specific oligonucleotide aptamer. In addition, AcLDL exhibited a higher affinity for HEK cells transfected with nucleolin than those without. Further, intracellular accumulation of AcLDL was also inhibited by anti-nucleolin antibody. The results of this study suggest that nucleolin expressed on macrophages is a receptor for AcLDL. PMID:26328500

  12. Behavioral effects of a synthetic agonist selective for nociceptin/orphanin FQ peptide receptors in monkeys.

    PubMed

    Ko, Mei-Chuan; Woods, James H; Fantegrossi, William E; Galuska, Chad M; Wichmann, Jürgen; Prinssen, Eric P

    2009-08-01

    Behavioral effects of a nonpeptidic NOP (nociceptin/orphanin FQ Peptide) receptor agonist, Ro 64-6198, have not been studied in primate species. The aim of the study was to verify the receptor mechanism underlying the behavioral effects of Ro 64-6198 and to systematically compare behavioral effects of Ro 64-6198 with those of a mu-opioid receptor agonist, alfentanil, in monkeys. Both Ro 64-6198 (0.001-0.06 mg/kg, s.c.) and alfentanil (0.001-0.06 mg/kg, s.c.) produced antinociception against an acute noxious stimulus (50 degrees C water) and capsaicin-induced allodynia. An NOP receptor antagonist, J-113397 (0.01-0.1 mg/kg, s.c.), dose-dependently produced rightward shifts of the dose-response curve of Ro 64-6198-induced antinociception. The apparent pA(2) value of J-113397 was 8.0. Antagonist studies using J-113397 and naltrexone revealed that Ro 64-6198 produced NOP receptor-mediated antinociception independent of mu-opioid receptors. In addition, alfentanil dose-dependently produced respiratory depression and itch/scratching responses, but antinociceptive doses of Ro 64-6198 did not produce such effects. More important, Ro 64-6198 did not produce reinforcing effects comparable with those of alfentanil, cocaine, or methohexital under self-administration procedures in monkeys. These results provide the first functional evidence that the activation of NOP receptors produces antinociception without reinforcing effects in primates. Non-peptidic NOP receptor agonists may have therapeutic value as novel analgesics without abuse liability in humans. PMID:19279568

  13. The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse

    PubMed Central

    Gu, Zhenyu; Nomura, Masatoshi; Simpson, Brenda B.; Lei, Hong; Feijen, Alie; van den Eijnden-van Raaij, Janny; Donahoe, Patricia K.; Li, En

    1998-01-01

    ActRIB is a type I transmembrane serine/threonine kinase receptor that has been shown to form heteromeric complexes with the type II activin receptors to mediate activin signal. To investigate the function of ActRIB in mammalian development, we generated ActRIB-deficient ES cell lines and mice by gene targeting. Analysis of the ActRIB−/− embryos showed that the epiblast and the extraembryonic ectoderm were disorganized, resulting in disruption and developmental arrest of the egg cylinder before gastrulation. To assess the function of ActRIB in mesoderm formation and gastrulation, chimera analysis was conducted. We found that ActRIB−/− ES cells injected into wild-type blastocysts were able to contribute to the mesoderm in chimeric embryos, suggesting that ActRIB is not required for mesoderm formation. Primitive streak formation, however, was impaired in chimeras when ActRIB−/− cells contributed highly to the epiblast. Further, chimeras generated by injection of wild-type ES cells into ActRIB−/− blastocysts formed relatively normal extraembryonic tissues, but the embryo proper developed poorly probably resulting from severe gastrulation defect. These results provide genetic evidence that ActRIB functions in both epiblast and extraembryonic cells to mediate signals that are required for egg cylinder organization and gastrulation. PMID:9512518

  14. New, potent, selective, and short-acting peptidic V1a receptor agonists.

    PubMed

    Wisniewski, Kazimierz; Galyean, Robert; Tariga, Hiroe; Alagarsamy, Sudarkodi; Croston, Glenn; Heitzmann, Joshua; Kohan, Arash; Wisniewska, Halina; Laporte, Régent; Rivière, Pierre J-M; Schteingart, Claudio D

    2011-07-14

    [Arg(8)]vasopressin (AVP) produces vasoconstriction via V(1a) receptor (V(1a)R)-mediated vascular smooth muscle cell contraction and is being used to increase blood pressure in septic shock, a form of vasodilatory hypotension. However, AVP also induces V(2) receptor (V(2)R)-mediated antidiuresis, vasodilation, and coagulation factor release, all deleterious in septic shock. The V(1a)R agonist terlipressin (H-Gly(3)[Lys(8)]VP) also lacks selectivity vs the V(2)R and has sizably longer duration of action than AVP, preventing rapid titration of its vasopressor effect in the clinic. We designed and synthesized new short acting V(1a)R selective analogues of general structure [Xaa(2),Ile(3),Yaa(4),Zaa(8)]VP. The most potent and selective compounds in in vitro functional assays (e.g., [Phe(2),Ile(3),Asn(Me(2))(4),Orn(8)]VP (31), [Phe(2),Ile(3),Asn((CH(2))(3)OH)(4),Orn(8)]VP (34), [Phe(2),Ile(3),Hgn(4),Orn(iPr)(8)]VP (45), [Phe(2),Ile(3),Asn(Et)(4),Dab(8)]VP (49), [Thi(2),Ile(3),Orn(iPr)(8)]VP (59), [Cha(2),Ile(3),Asn(4),Orn(iPr)(8)]VP (68)) were tested by intravenous bolus in rats for duration of vasopressive action. Analogues 31, 34, 45, and 49 were as short-acting as AVP. Compound 45, FE 202158, is currently undergoing clinical trials in septic shock. PMID:21688787

  15. A [3]rotaxane with two porphyrinic plates acting as an adaptable receptor.

    PubMed

    Frey, Julien; Tock, Christian; Collin, Jean-Paul; Heitz, Valérie; Sauvage, Jean-Pierre

    2008-04-01

    Following a multistep procedure, the copper(I)-templated strategy allowed preparation of a multifunctional [3]rotaxane. The dumbbell consists of a central two-bidentate chelate unit and two terminal stoppers. The two rings threaded on the rotaxane axis consist each of a 1,10-phenanthroline-incorporating macrocycle, rigidly connected to an appended zinc-complexed porphyrin. The copper(I) template can be removed, affording a free rotaxane whose two rings can glide freely along the axis and spin around it. The dumbbell being very long (approximately 85 A in its extended conformation from one stopper to the other), the porphyrin-porphyrin distance can be varied over a wide range. The two porphyrinic plates constitute the key elements of a receptor able to complex various guests between the plates. The ability of the threaded rings to move freely makes the host perfectly adjustable, allowing capture of geometrically very different guests. The copper(I)-complexed rotaxane also acts as an efficient receptor, although its adaptability is obviously more limited than that of its free rotaxane counterpart. PMID:18338892

  16. The orphan nuclear receptor DAX-1 acts as a novel transcriptional corepressor of PPAR{gamma}

    SciTech Connect

    Kim, Gwang Sik; Lee, Gha Young; Nedumaran, Balachandar; Park, Yun-Yong; Kim, Kyung Tae; Park, Sang Chul; Lee, Young Chul; Kim, Jae Bum Choi, Hueng-Sik

    2008-05-30

    DAX-1 is an atypical nuclear receptor (NR) which functions primarily as a transcriptional corepressor of other NRs via heterodimerization. Peroxisome proliferator-activated receptor (PPAR) {gamma} is a ligand-dependent NR which performs a key function in adipogenesis. In this study, we evaluated a novel cross-talk mechanism between DAX-1 and PPAR{gamma}. Transient transfection assays demonstrated that DAX-1 inhibits the transactivity of PPAR{gamma} in a dose-dependent manner. DAX-1 directly competed with the PPAR{gamma} coactivator (PGC)-1{alpha} for binding to PPAR{gamma}. Endogenous levels of DAX-1 were significantly lower in differentiated 3T3-L1 adipocytes as compared to preadipocytes. Using a retroviral expression system, we demonstrated that DAX-1 overexpression downregulates the expression of PPAR{gamma} target genes, resulting in an attenuation of adipogenesis in 3T3-L1 cells. Our results suggest that DAX-1 acts as a corepressor of PPAR{gamma} and performs a potential function in the regulation of PPAR{gamma}-mediated cellular differentiation.

  17. Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor.

    PubMed

    Dinan, L; Whiting, P; Girault, J P; Lafont, R; Dhadialla, T S; Cress, D E; Mugat, B; Antoniewski, C; Lepesant, J A

    1997-11-01

    Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 microM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 microM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Delta23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity. PMID:9581538

  18. Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor.

    PubMed Central

    Dinan, L; Whiting, P; Girault, J P; Lafont, R; Dhadialla, T S; Cress, D E; Mugat, B; Antoniewski, C; Lepesant, J A

    1997-01-01

    Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 microM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 microM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Delta23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity. PMID:9581538

  19. Histamine influences body temperature by acting at H1 and H3 receptors on distinct populations of preoptic neurons

    PubMed Central

    Lundius, Ebba Gregorsson; Sanchez-Alavez, Manuel; Ghochani, Yasmin; Klaus, Joseph; Tabarean, Iustin V.

    2010-01-01

    The preoptic area/anterior hypothalamus (PO/AH), a region that contains neurons that control thermoregulation, is the main locus at which histamine affects body temperature. Here we report that histamine reduced the spontaneous firing rate of GABAergic preoptic neurons by activating H3 subtype histamine receptors. This effect involved a decrease in the level of phosphorylation of the extracellular signal-regulated kinase (ERK) and was not dependent on synaptic activity. Furthermore, a population of nonGABAergic neurons was depolarized and their firing rate was enhanced by histamine acting at H1 subtype receptors. In our experiments, activation of the H1R receptors was linked to the PLC pathway and Ca2+ release from intracellular stores. This depolarization persisted in TTX or when fast synaptic potentials were blocked indicating that it represents a postsynaptic effect. Single-cell reverse transcription –PCR analysis revealed expression of H3 receptors in a population of GABAergic neurons while H1 receptors were expressed in nonGABAergic cells. Histamine applied in the median preoptic nucleus induced a robust, long lasting hyperthermia effect that was mimicked by either H1 or H3 histamine receptor subtype specific agonists. Our data indicate that histamine modulates the core body temperature by acting at two distinct populations of preoptic neurons which express H1 and H3 receptor subtypes, respectively. PMID:20335473

  20. Histamine influences body temperature by acting at H1 and H3 receptors on distinct populations of preoptic neurons.

    PubMed

    Lundius, Ebba Gregorsson; Sanchez-Alavez, Manuel; Ghochani, Yasmin; Klaus, Joseph; Tabarean, Iustin V

    2010-03-24

    The preoptic area/anterior hypothalamus, a region that contains neurons that control thermoregulation, is the main locus at which histamine affects body temperature. Here we report that histamine reduced the spontaneous firing rate of GABAergic preoptic neurons by activating H3 subtype histamine receptors. This effect involved a decrease in the level of phosphorylation of the extracellular signal-regulated kinase and was not dependent on synaptic activity. Furthermore, a population of non-GABAergic neurons was depolarized, and their firing rate was enhanced by histamine acting at H1 subtype receptors. In our experiments, activation of the H1R receptors was linked to the PLC pathway and Ca(2+) release from intracellular stores. This depolarization persisted in TTX or when fast synaptic potentials were blocked, indicating that it represents a postsynaptic effect. Single-cell reverse transcription-PCR analysis revealed expression of H3 receptors in a population of GABAergic neurons, while H1 receptors were expressed in non-GABAergic cells. Histamine applied in the median preoptic nucleus induced a robust, long-lasting hyperthermia effect that was mimicked by either H1 or H3 histamine receptor subtype-specific agonists. Our data indicate that histamine modulates the core body temperature by acting at two distinct populations of preoptic neurons that express H1 and H3 receptor subtypes, respectively. PMID:20335473

  1. Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

    PubMed

    Remmers, A E; Clark, M J; Alt, A; Medzihradsky, F; Woods, J H; Traynor, J R

    2000-05-19

    The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane. PMID:10822058

  2. Rho/ROCK acts downstream of lysophosphatidic acid receptor 1 in modulating P2X3 receptor-mediated bone cancer pain in rats

    PubMed Central

    Wu, Jing-xiang; Yuan, Xiao-min; Wang, Qiong; Wei, Wang

    2016-01-01

    Background Lysophosphatidic acid receptor 1 and Rho/ROCK signaling is implicated in bone cancer pain development. However, it remains unknown whether the two signaling pathways function together in P2X3 receptor-mediated bone cancer pain. Results In this study, using a rat model of bone cancer, we examined the expression of P2X3 and lysophosphatidic acid receptor 1 in rat dorsal root ganglion neurons and further dissected whether lysophosphatidic acid receptor 1 and Rho/ROCK-mediated pathways interacted in modulating rat pain behavior. Bone cancer was established by inoculating Walker 256 cells into the left tibia of female Wistar rats. We observed a gradual and yet significant decline in mean paw withdrawal threshold in rats with bone cancer, but not in control rats. Our immunohistochemical staining revealed that the number of P2X3- and lysophosphatidic acid receptor 1-positive dorsal root ganglion neurons was significantly greater in rats with bone cancer than control rats. Lysophosphatidic acid receptor 1 blockade with VPC32183 significantly attenuated decline in mean paw withdrawal threshold. Flinching behavior test further showed that lysophosphatidic acid receptor 1 inhibition with VPC32183 transiently but significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Rho inhibition by intrathecal BoTXC3 caused a rapid reversal in decline in mean paw withdrawal threshold of rats with bone cancer. Flinching behavior test showed that BoTXC3 transiently and significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Similar findings were observed with ROCK inhibition by intrathecal Y27632. Furthermore, VPC32183 and BoTXC3 effectively aborted the appearance of lysophosphatidic acid-induced calcium influx peak. Conclusions Lysophosphatidic acid and its receptor LPAR1, acting through the Rho-ROCK pathway, regulate P2X3 receptor in the development of both mechanical and spontaneous pain in bone cancer. PMID:27094551

  3. Scalable Production of Highly Sensitive Nanosensors Based on Graphene Functionalized with a Designed G Protein-Coupled Receptor

    NASA Astrophysics Data System (ADS)

    Lerner, Mitchell B.; Matsunaga, Felipe; Han, Gang Hee; Hong, Sung Ju; Xi, Jin; Crook, Alexander; Perez-Aguilar, Jose Manuel; Park, Yung Woo; Saven, Jeffery G.; Liu, Renyu; Johnson, A. T. Charlie

    2014-05-01

    We have developed a novel, all-electronic biosensor for opioids that consists of an engineered mu opioid receptor protein, with high binding affinity for opioids, chemically bonded to a graphene field-effect transistor to read out ligand binding. A variant of the receptor protein that provided chemical recognition was computationally redesigned to enhance its solubility and stability in an aqueous environment. A shadow mask process was developed to fabricate arrays of hundreds of graphene transistors with average mobility of ~1500 cm2 V-1 s-1 and yield exceeding 98%. The biosensor exhibits high sensitivity and selectivity for the target naltrexone, an opioid receptor antagonist, with a detection limit of 10 pg/mL.

  4. Neurogenic contractions in intraocular porcine ciliary arteries are mediated by α₂-adrenoceptors and NPY₁ receptors and are inhibited by prostaglandin E₂ acting on prejunctional EP₄ receptors.

    PubMed

    Kringelholt, Sidse; Simonsen, Ulf; Bek, Toke

    2013-02-01

    Prostaglandin analogues and adrenergic drugs are used to reduce the intraocular pressure in glaucoma, which may partly be due to an effect on the tone of the intraocular arteries supplying the ciliary body. The aim of the present study was to investigate the interaction between prostaglandins and autonomic nervous activity induced by electrical stimulation of the tone in these ciliary vessels. The intraocular part of porcine ciliary arteries were isolated and mounted in a microvascular myograph for isometric tension recordings, and the effect of prostaglandin E(2) on electrically induced contractions was studied in the presence of selective EP receptor antagonists. PGE(2) induced concentration-dependent inhibition of electrically induced contractions of intraocular ciliary arteries which depended on the presence of the vascular endothelium. The effect of PGE(2) was blocked by an EP(4) receptor antagonist but not by an EP(1) receptor antagonist. The neurogenic contractions were partially inhibited by an α(2)-adrenoceptor antagonist and totally inhibited by a NPY(1) receptor antagonist. The effect of these antagonists was similar when contraction was induced by noradrenaline and NPY. Neurogenic contractions in intraocular porcine arteries are mediated by α(2)-adrenoceptors and NPY(1) receptors and can be inhibited by PGE(2) acting on prejunctional EP(4) receptors. This contributes to a further understanding of the role of the autonomic nervous system and prostaglandins for regulating blood flow to the anterior segment of the eye. PMID:23178872

  5. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors.

    PubMed

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-04-12

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially. PMID:26967251

  6. Central kappa opioid receptor-evoked changes in renal function in conscious rats: participation of renal nerves.

    PubMed

    Kapusta, D R; Obih, J C

    1993-10-01

    The present investigations examined the cardiovascular and renal responses produced by central nervous system stimulation of kappa opioid receptors by the selective kappa opioid receptor agonist, U-50488H, in conscious Sprague-Dawley rats. Administration of U-50488H (1 microgram total) into the lateral cerebroventricle produced a profound diuretic and antinatriuretic response. In addition, concurrent with the decrease in urinary sodium excretion, i.c.v. U-50488H elicited an increase in renal sympathetic nerve activity. The increases in urine flow rate and renal sympathetic nerve activity and the decrease in urinary sodium excretion produced by U-50488H were completely prevented in rats that had undergone pretreatment with the selective kappa opioid receptor antagonist, nor-binaltorphimine. In contrast, in animals that had undergone irreversible mu opioid receptor blockade with the selective mu opioid receptor antagonist, beta-funaltrexamine, central U-50488H administration elicited similar diuretic and antinatriuretic responses as observed in intact naive animals. In further studies, the antinatriuretic response produced by i.c.v. U-50488H was completely abolished in rats that had undergone chronic bilateral renal denervation, a technique used to remove the influence of the renal sympathetic nerves. Glomerular filtration rates and effective renal plasma flows were not altered by i.c.v. administration of U-50488H in intact or renal denervated animals. Together, these studies provide evidence for the role of central kappa opioid receptor mechanisms in the regulation of urinary sodium and water excretion. Moreover, these studies indicate that the changes in renal sodium handling produced by central kappa opioid agonists result from an action of these compounds to modulate sympathetic neural outflow to the kidneys. PMID:8229746

  7. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors

    PubMed Central

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-01-01

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially. PMID:26967251

  8. CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

    PubMed Central

    Mamedova, Liaman; Capra, Valérie; Accomazzo, Maria Rosa; Gao, Zhan-Guo; Ferrario, Silvia; Fumagalli, Marta; Abbracchio, Maria P.; Rovati, G. Enrico; Jacobson, Kenneth A.

    2016-01-01

    Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y1,2,4,6,11,12,13,14 receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 μM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 μM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y1,2,4,6 receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 μM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 μM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions. PMID:16280122

  9. Sweet Taste-Sensing Receptors Expressed in Pancreatic β-Cells: Sweet Molecules Act as Biased Agonists

    PubMed Central

    Nakagawa, Yuko; Ohtsu, Yoshiaki; Medina, Anya; Nagasawa, Masahiro

    2014-01-01

    The sweet taste receptors present in the taste buds are heterodimers comprised of T1R2 and T1R3. This receptor is also expressed in pancreatic β-cells. When the expression of receptor subunits is determined in β-cells by quantitative reverse transcription polymerase chain reaction, the mRNA expression level of T1R2 is extremely low compared to that of T1R3. In fact, the expression of T1R2 is undetectable at the protein level. Furthermore, knockdown of T1R2 does not affect the effect of sweet molecules, whereas knockdown of T1R3 markedly attenuates the effect of sweet molecules. Consequently, a homodimer of T1R3 functions as a receptor sensing sweet molecules in β-cells, which we designate as sweet taste-sensing receptors (STSRs). Various sweet molecules activate STSR in β-cells and augment insulin secretion. With regard to intracellular signals, sweet molecules act on STSRs and increase cytoplasmic Ca2+ and/or cyclic AMP (cAMP). Specifically, when an STSR is stimulated by one of four different sweet molecules (sucralose, acesulfame potassium, sodium saccharin, or glycyrrhizin), distinct signaling pathways are activated. Patterns of changes in cytoplasmic Ca2+ and/or cAMP induced by these sweet molecules are all different from each other. Hence, sweet molecules activate STSRs by acting as biased agonists. PMID:24741449

  10. Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.

    PubMed

    Chaturvedi, K; Bandari, P; Chinen, N; Howells, R D

    2001-04-13

    This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors. PMID:11152677

  11. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

    PubMed

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H

    2013-12-01

    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors. PMID:23884575

  12. Role of kappa and delta opioid receptors in mediating morphine-induced antinociception in morphine tolerant infant rats

    PubMed Central

    Stoller, Dawn C.; Sim-Selley, Laura J.; Smith, Forrest L.

    2011-01-01

    We have previously noted that the antinociceptive efficacy of morphine was significantly decreased in rat pups chronically infused with morphine from implanted osmotic minipumps. In this study, morphine was fully efficacious (i.e., 100% maximum possible effect, %MPE) in the 52 ºC tail-immersion test after a 72-h infusion from implanted saline-filled osmotic minipumps. However, administration of up to 1000 mg/kg s.c. morphine failed to elicit greater than a 27% MPE in rats infused with morphine at 2 mg/kg/h. Morphine was more efficacious when the water bath temperature was decreased to 49 ºC. Experiments were conducted to determine the mechanisms whereby chronic morphine administration leads to a decrease in antinociceptive efficacy. The kappa-opioid antagonist nor-binalorphimine completely blocked the antinociceptive effects of morphine in morphine-infused rat pups. The kappa agonist U50,488 elicited antinociception however, the requirement to use higher doses in morphine- than saline-infused rats indicates that kappa cross-tolerance was present. Thus, in tolerant rats the antinociceptive effects of high doses of morphine appear to be mediated through kappa-opioid receptors. The delta-opioid antagonist naltrindole was inactive in both treatment groups. DAMGO-stimulated [35S]GTPγS and [3H]naloxone binding reveal that the anatomical distribution of the mu-opioid receptor was consistent with that of the adult rat brain. In adult rats, the mu-opioid receptor is desensitized during morphine tolerance. However, desensitization was not evident in P17 rats based on the lack of significant decreases in [35S]GTPγS binding. Furthermore, [3H]naloxone binding indicated a lack of mu receptor downregulation in morphine-tolerant rat pups. PMID:17300766

  13. BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice.

    PubMed

    Watanabe, Kenta; Hirata, Michiko; Tominari, Tsukasa; Matsumoto, Chiho; Endo, Yasuyuki; Murphy, Gillian; Nagase, Hideaki; Inada, Masaki; Miyaura, Chisato

    2016-09-01

    Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERβ. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male. PMID:27402268

  14. Structural features of phenoxycarbonylimino neonicotinoids acting at the insect nicotinic receptor.

    PubMed

    Ohno, Ikuya; Tomizawa, Motohiro; Miyazu, Nozomi; Kushibiki, Gohito; Noda, Kumiko; Hasebe, Yasunori; Durkin, Kathleen A; Miyake, Taiji; Kagabu, Shinzo

    2010-10-01

    Substituted-phenoxycarbonylimino neonicotinoid ligands with an electron-donating group showed significantly higher affinity to the insect nicotinic receptor relative to that of the analogue with an electron-withdrawing substituent, thereby establishing in silico binding site interaction model featuring that the phenoxy ring of neonicotinoids and the receptor loop D tryptophan indole plane form a face-to-edge aromatic interaction. PMID:20729079

  15. The 37kDa/67kDa Laminin Receptor acts as a receptor for Aβ42 internalization

    PubMed Central

    Da Costa Dias, Bianca; Jovanovic, Katarina; Gonsalves, Danielle; Moodley, Kiashanee; Reusch, Uwe; Knackmuss, Stefan; Weinberg, Marc S.; Little, Melvyn; Weiss, Stefan F. T.

    2014-01-01

    Neuronal loss is a major neuropathological hallmark of Alzheimer's disease (AD). The associations between soluble Aβ oligomers and cellular components cause this neurotoxicity. The 37 kDa/67 kDa laminin receptor (LRP/LR) has recently been implicated in Aβ pathogenesis. In this study the mechanism underlying the pathological role of LRP/LR was elucidated. Försters Resonance Energy Transfer (FRET) revealed that LRP/LR and Aβ form a biologically relevant interaction. The ability of LRP/LR to form stable associations with endogenously shed Aβ was confirmed by pull down assays and Aβ-ELISAs. Antibody blockade of this association significantly lowered Aβ42 induced apoptosis. Furthermore, antibody blockade and shRNA mediated downregulation of LRP/LR significantly hampered Aβ42 internalization. These results suggest that LRP/LR is a receptor for Aβ42 internalization, mediating its endocytosis and contributing to the cytotoxicity of the neuropeptide by facilitating intra-cellular Aβ42 accumulation. These findings recommend anti-LRP/LR specific antibodies and shRNAs as potential therapeutic tools for AD treatment. PMID:24990253

  16. Prostanoids regulate angiogenesis acting primarily on IP and EP4 receptors.

    PubMed

    Hoang, Khuyen Gia; Allison, Sarah; Murray, Michael; Petrovic, Nenad

    2015-09-01

    Angiogenesis is regulated by numerous activators and inhibitors, including prostanoids. Although many studies have identified their roles in inflammation, regulatory functions of prostanoids in angiogenesis are poorly understood. Here, we compared the activation of angiogenesis in vitro by two prostanoids with important vascular roles: prostaglandin E2 (PGE2) - thought to be the most important prostanoid activator of angiogenesis - and prostaglandin I2 (prostacyclin or PGI2), whose receptors are predominantly expressed in endothelial cells. Both of these prostanoids activate G-protein coupled receptors: EP1, EP2, EP3 and EP4 by PGE2 and IP by prostacyclin. Human umbilical vein endothelial cells (HUVECs) were used to characterize two pivotal pro-angiogenic processes in vitro: cell migration (using the matrigel droplet assay developed in our laboratory) and "tube formation" (a widely accepted method of assessing formation of blood vessel precursors). The suppression of cell migration and tube formation by the IP-specific antagonist CAY10441 was more extensive (~80%) than by the EP4-specific antagonist L-161,982 (~20%). AH6809, an antagonist of EP1, EP2 and EP3 receptors did not significantly suppress angiogenesis. Expression of the pro-angiogenic receptors KDR and Tie-2 in HUVECs was preferentially suppressed by antagonism of IP and EP4 receptors, respectively. EP4 and IP receptor agonists elicited biphasic actions on angiogenic processes in which there was activation at low concentration, and rapid desensitization at high concentrations - a characteristic common to many G-protein coupled receptors. Together these findings suggest that the prostacyclin-IP pathway plays a major role in the regulation of pro-angiogenic processes in HUVECs. PMID:26188701

  17. Antiandrogens Act as Selective Androgen Receptor Modulators at the Proteome Level in Prostate Cancer Cells*

    PubMed Central

    Brooke, Greg N.; Gamble, Simon C.; Hough, Michael A.; Begum, Shajna; Dart, D. Alwyn; Odontiadis, Michael; Powell, Sue M.; Fioretti, Flavia M.; Bryan, Rosie A.; Waxman, Jonathan; Wait, Robin; Bevan, Charlotte L.

    2015-01-01

    Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic

  18. Different Pathways Act Downstream of the CEP Peptide Receptor CRA2 to Regulate Lateral Root and Nodule Development.

    PubMed

    Mohd-Radzman, Nadiatul A; Laffont, Carole; Ivanovici, Ariel; Patel, Neha; Reid, Dugald; Stougaard, Jens; Frugier, Florian; Imin, Nijat; Djordjevic, Michael A

    2016-08-01

    C-TERMINALLY ENCODED PEPTIDEs (CEPs) control root system architecture in a non-cell-autonomous manner. In Medicago truncatula, MtCEP1 affects root development by increasing nodule formation and inhibiting lateral root emergence by unknown pathways. Here, we show that the MtCEP1 peptide-dependent increase in nodulation requires the symbiotic signaling pathway and ETHYLENE INSENSITIVE2 (EIN2)/SICKLE (SKL), but acts independently of SUPER NUMERIC NODULES. MtCEP1-dependent inhibition of lateral root development acts through an EIN2-independent mechanism. MtCEP1 increases nodulation by promoting rhizobial infections, the developmental competency of roots for nodulation, the formation of fused nodules, and an increase in frequency of nodule development that initiates at proto-phloem poles. These phenotypes are similar to those of the ein2/skl mutant and support that MtCEP1 modulates EIN2-dependent symbiotic responses. Accordingly, MtCEP1 counteracts the reduction in nodulation induced by increasing ethylene precursor concentrations, and an ethylene synthesis inhibitor treatment antagonizes MtCEP1 root phenotypes. MtCEP1 also inhibits the development of EIN2-dependent pseudonodule formation. Finally, mutants affecting the COMPACT ROOT ARCHITECTURE2 (CRA2) receptor, which is closely related to the Arabidopsis CEP Receptor1, are unresponsive to MtCEP1 effects on lateral root and nodule formation, suggesting that CRA2 is a CEP peptide receptor mediating both organogenesis programs. In addition, an ethylene inhibitor treatment counteracts the cra2 nodulation phenotype. These results indicate that MtCEP1 and its likely receptor, CRA2, mediate nodulation and lateral root development through different pathways. PMID:27342310

  19. Different Pathways Act Downstream of the CEP Peptide Receptor CRA2 to Regulate Lateral Root and Nodule Development1[OPEN

    PubMed Central

    Mohd-Radzman, Nadiatul A.; Ivanovici, Ariel; Frugier, Florian; Djordjevic, Michael A.

    2016-01-01

    C-TERMINALLY ENCODED PEPTIDEs (CEPs) control root system architecture in a non-cell-autonomous manner. In Medicago truncatula, MtCEP1 affects root development by increasing nodule formation and inhibiting lateral root emergence by unknown pathways. Here, we show that the MtCEP1 peptide-dependent increase in nodulation requires the symbiotic signaling pathway and ETHYLENE INSENSITIVE2 (EIN2)/SICKLE (SKL), but acts independently of SUPER NUMERIC NODULES. MtCEP1-dependent inhibition of lateral root development acts through an EIN2-independent mechanism. MtCEP1 increases nodulation by promoting rhizobial infections, the developmental competency of roots for nodulation, the formation of fused nodules, and an increase in frequency of nodule development that initiates at proto-phloem poles. These phenotypes are similar to those of the ein2/skl mutant and support that MtCEP1 modulates EIN2-dependent symbiotic responses. Accordingly, MtCEP1 counteracts the reduction in nodulation induced by increasing ethylene precursor concentrations, and an ethylene synthesis inhibitor treatment antagonizes MtCEP1 root phenotypes. MtCEP1 also inhibits the development of EIN2-dependent pseudonodule formation. Finally, mutants affecting the COMPACT ROOT ARCHITECTURE2 (CRA2) receptor, which is closely related to the Arabidopsis CEP Receptor1, are unresponsive to MtCEP1 effects on lateral root and nodule formation, suggesting that CRA2 is a CEP peptide receptor mediating both organogenesis programs. In addition, an ethylene inhibitor treatment counteracts the cra2 nodulation phenotype. These results indicate that MtCEP1 and its likely receptor, CRA2, mediate nodulation and lateral root development through different pathways. PMID:27342310

  20. Aldose Reductase acts as a Selective Derepressor of PPARγ and Retinoic Acid Receptor

    PubMed Central

    Thiagarajan, Devi; Ananthakrishnan, Radha; Zhang, Jinghua; O’Shea, Karen M.; Quadri, Nosirudeen; Li, Qing; Sas, Kelli; Jing, Xiao; Rosario, Rosa; Pennathur, Subramaniam; Schmidt, Ann Marie; Ramasamy, Ravichandran

    2016-01-01

    Summary Histone deacetylase 3 (HDAC3), a chromatin modifying enzyme, requires association with the deacetylase containing domain (DAD) of the nuclear receptor co-repressors NCOR1 and SMRT for its stability and activity. Here we show that aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, competes with HDAC3 to bind the NCOR1/SMRT DAD. Increased AR expression leads to HDAC3 degradation followed by increased PPARγ signaling resulting in lipid accumulation in the heart. AR also downregulates expression of nuclear corepressor complex cofactors including Gps2 and Tblr1, thus affecting activity of the nuclear corepressor complex itself. Though AR reduces HDAC3-corepressor complex formation, it specifically de-represses the retinoic acid receptor (RAR), but not other nuclear receptors such as the thyroid receptor (TR) and liver X receptor (LXR). In summary, this work defines a distinct role for AR in lipid and retinoid metabolism through HDAC3 regulation and consequent de-repression of PPARγ and RAR. PMID:27052179

  1. Abscisic Acid Analogues That Act as Universal or Selective Antagonists of Phytohormone Receptors.

    PubMed

    Rajagopalan, Nandhakishore; Nelson, Ken M; Douglas, Amy F; Jheengut, Vishal; Alarcon, Idralyn Q; McKenna, Sean A; Surpin, Marci; Loewen, Michele C; Abrams, Suzanne R

    2016-09-13

    The plant hormone abscisic acid (ABA) plays many important roles in controlling plant development and physiology, from flowering to senescence. ABA is now known to exert its effects through a family of soluble ABA receptors, which in Arabidopsis thaliana has 13 members divided into three clades. Homologues of these receptors are present in other plants, also in relatively large numbers. Investigation of the roles of each homologue in mediating the diverse physiological roles of ABA is hampered by this genetic redundancy. We report herein the in vitro screening of a targeted ABA-like analogue library and identification of novel antagonist hits, including the analogue PBI686 that had been developed previously as a probe for identifying ABA-binding proteins. Further in vitro characterization of PBI686 and development of second-generation leads yielded both receptor-selective and universal antagonist hits. In planta assays in different species have demonstrated that these antagonist leads can overcome various ABA-induced physiological changes. While the general antagonists open up a hitherto unexplored avenue for controlling plant growth through inhibition of ABA-regulated physiological processes, the receptor-selective antagonist can be developed into chemical probes to explore the physiological roles of individual receptors. PMID:27523384

  2. Transmembrane chemokines act as receptors in a novel mechanism termed inverse signaling

    PubMed Central

    Hattermann, Kirsten; Gebhardt, Henrike; Krossa, Sebastian; Ludwig, Andreas; Lucius, Ralph

    2016-01-01

    The transmembrane chemokines CX3CL1/fractalkine and CXCL16 are widely expressed in different types of tumors, often without an appropriate expression of their classical receptors. We observed that receptor-negative cancer cells could be stimulated by the soluble chemokines. Searching for alternative receptors we detected that all cells expressing or transfected with transmembrane chemokine ligands bound the soluble chemokines with high affinity and responded by phosphorylation of intracellular kinases, enhanced proliferation and anti-apoptosis. This activity requires the intracellular domain and apparently the dimerization of the transmembrane chemokine ligand. Thus, shed soluble chemokines can generate auto- or paracrine signals by binding and activating their transmembrane forms. We term this novel mechanism “inverse signaling”. We suppose that inverse signaling is an autocrine feedback and fine-tuning system in the communication between cells that in tumors supports stabilization and proliferation. DOI: http://dx.doi.org/10.7554/eLife.10820.001 PMID:26796342

  3. ATP appears to act via different receptors in terminals vs. somata of the Hypothalamic Neurohypophysial System

    PubMed Central

    Knott, Thomas K.; Hussy, Nicolas; Cuadra, Adolfo E.; Lee, Ryan H.; Ortiz-Miranda, Sonia; Custer, Edward E.; Lemos, José R.

    2012-01-01

    ATP-induced ionic currents were investigated in isolated terminals and somata of the Hypothalamic Neurohypophysial System (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence; EC50 of 50 μM in somata vs. 9.6 μM in terminals. The ATP induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a P2X receptor. However, the suramin inhibition was significantly different in the two HNS compartments: IC50 of 3.6 μM in somata vs 11.6 μM in terminals. Also, both HNS compartments show significantly different responses to the purinergic receptor agonists ATP-γ-S and Benzoyl-benzoyl-ATP. Finally, there was an initial desensitization to ATP upon successive stimulations in the terminals which was not observed in the somata. These differences in EC50, inactivation, desensitization, and agonist sensitivity in terminals vs. somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurons. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4, and 7 but not 6 have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and OT terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals vs. somata of these CNS neurons. PMID:22340013

  4. Desensitization of G protein-coupled receptors and neuronal functions.

    PubMed

    Gainetdinov, Raul R; Premont, Richard T; Bohn, Laura M; Lefkowitz, Robert J; Caron, Marc G

    2004-01-01

    G protein-coupled receptors (GPCRs) have proven to be the most highly favorable class of drug targets in modern pharmacology. Over 90% of nonsensory GPCRs are expressed in the brain, where they play important roles in numerous neuronal functions. GPCRs can be desensitized following activation by agonists by becoming phosphorylated by members of the family of G protein-coupled receptor kinases (GRKs). Phosphorylated receptors are then bound by arrestins, which prevent further stimulation of G proteins and downstream signaling pathways. Discussed in this review are recent progress in understanding basics of GPCR desensitization, novel functional roles, patterns of brain expression, and receptor specificity of GRKs and beta arrestins in major brain functions. In particular, screening of genetically modified mice lacking individual GRKs or beta arrestins for alterations in behavioral and biochemical responses to cocaine and morphine has revealed a functional specificity in dopamine and mu-opioid receptor regulation of locomotion and analgesia. An important and specific role of GRKs and beta arrestins in regulating physiological responsiveness to psychostimulants and morphine suggests potential involvement of these molecules in certain brain disorders, such as addiction, Parkinson's disease, mood disorders, and schizophrenia. Furthermore, the utility of a pharmacological strategy aimed at targeting this GPCR desensitization machinery to regulate brain functions can be envisaged. PMID:15217328

  5. l-Isocorypalmine reduces behavioral sensitization and rewarding effects of cocaine in mice by acting on dopamine receptors*

    PubMed Central

    Xu, Wei; Wang, Yujun; Ma, Zhongze; Chiu, Yi-Ting; Huang, Peng; Rasakham, Khampaseuth; Unterwald, Ellen; Lee, David Y.-W.; Liu-Chen, Lee-Yuan

    2013-01-01

    Background We previously reported isolation of l-isocorypalmine (l-ICP), a mono-demethylated analog of l-tetrahydropalmatine (l-THP), from the plant Corydalis yanhusuo. Here we characterized its in vitro pharmacological properties and examined its effects on cocaine-induced behaviors in mice. Methods Receptor binding, cAMP and [35S]GTPγS assays were used to examine pharmacological actions of l-ICP in vitro. Effects of I-ICP on cocaine-induced locomotor hyperactivity and sensitization and conditioned place preference (CPP) in mice were investigated. HPLC was employed to analyze metabolites of I-ICP in mouse serum. Results Among more than 40 targets screened, l-ICP and l-THP bound only to dopamine (DA) receptors. l-ICP was a high-affinity partial agonist of D1 and D5 receptors and a moderate-affinity antagonist of D2, D3 and D4 receptors, whereas l-THP bound to only D1 and D5 receptors, with lower affinities than l-ICP. At 10 mg/kg (i.p.), l-ICP inhibited spontaneous locomotor activity for a shorter time than l-THP. Pretreatment with l-ICP reduced cocaine-induced locomotor hyperactivities. Administration of l-ICP before cocaine once a day for 5 days reduced cocaine-induced locomotor sensitization on days 5 and 13 after 7 days of withdrawal. Pretreatment with l-ICP before cocaine daily for 6 days blocked cocaine-induced CPP, while l-ICP itself did not cause preference or aversion. HPLC analysis showed that l-ICP was the main compound in mouse serum following i.p. injection of l-ICP. Conclusions l-ICP likely acts as a D1 partial agonist and a D2 antagonist to produce its in vivo effects and may be a promising agent for treatment of cocaine addiction. PMID:24080315

  6. RAPID HETEROLOGOUS DESENSITIZATION OF ANTINOCICEPTIVE ACTIVITY BETWEEN MU OR DELTA OPIOID RECEPTORS AND CHEMOKINE RECEPTORS IN RATS

    PubMed Central

    Chen, Xiaohong; Geller, Ellen B.; Rogers, Thomas J.; Adler, Martin W.

    2007-01-01

    Previous studies have shown pretreatment with chemokines CCL5/RANTES (100 ng) or CXCL12/SDF-1alpha (100 ng) injected into the periaqueductal grey (PAG) region of the brain, 30 minutes (min) before the mu opioid agonist DAMGO (400 ng), blocked the antinociception induced by DAMGO in the in vivo cold water tail-flick (CWT) antinociceptive test in rats. In the present experiments, we tested whether the action of other agonists at mu and delta opioid receptors is blocked when CCL5/RANTES or CXCL12/SDF-1alpha is administered into the PAG 30 min before, or co-administered with, opioid agonists in the CWT assay. The results showed that (1) CXCL12/SDF-1alpha (100 ng, PAG) or CCL5/RANTES (100 ng, PAG), given 30 min before the opioid agonist morphine, or selective delta opioid receptor agonist DPDPE, blocked the antinociceptive effect of these drugs; (2) CXCL12/SDF-1alpha (100 ng, PAG) or CCL5/RANTES (100 ng, PAG), injected at the same time as DAMGO or DPDPE, significantly reduced the antinociceptive effect induced by these drugs. These results demonstrate that the heterologous desensitization is rapid between the mu or delta opioid receptors and either CCL5/RANTES receptor CCR5 or CXCL12/SDF-1alpha receptor CXCR4 in vivo, but the effect is greater if the chemokine is administered before the opioid. PMID:17049756

  7. Guanidino acids act as rho1 GABA(C) receptor antagonists.

    PubMed

    Chebib, Mary; Gavande, Navnath; Wong, Kit Yee; Park, Anna; Premoli, Isabella; Mewett, Kenneth N; Allan, Robin D; Duke, Rujee K; Johnston, Graham A R; Hanrahan, Jane R

    2009-10-01

    GABA(C) receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, beta-alanine and taurine were evaluated at human rho(1)GABA(C) receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-guanidinopropionic acid (IC(50) = 2.2 microM) and guanidinoacetic acid (IC(50) = 5.4 microM; K (B) = 7.75 microM [pK (B) = 5.11 +/- 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the beta-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure-activity-relationship profile required in developing novel therapeutic agents. PMID:19387831

  8. Cyclohexanol analogues are positive modulators of GABAA receptor currents and act as general anaesthetics in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    GABAA receptors meet all the pharmacological criteria required to be considered important general anaesthetic targets. In the following study, the modulatory effects of various commercially available and novel cyclohexanol were investigated on recombinant human '-aminobutyric acid (GABAA, a1ß2'2s) r...

  9. Development and Validation of Quantitative Structure-Activity Relationship Models for Compounds Acting on Serotoninergic Receptors

    PubMed Central

    Żydek, Grażyna; Brzezińska, Elżbieta

    2012-01-01

    A quantitative structure-activity relationship (QSAR) study has been made on 20 compounds with serotonin (5-HT) receptor affinity. Thin-layer chromatographic (TLC) data and physicochemical parameters were applied in this study. RP2 TLC 60F254 plates (silanized) impregnated with solutions of propionic acid, ethylbenzene, 4-ethylphenol, and propionamide (used as analogues of the key receptor amino acids) and their mixtures (denoted as S1–S7 biochromatographic models) were used in two developing phases as a model of drug-5-HT receptor interaction. The semiempirical method AM1 (HyperChem v. 7.0 program) and ACD/Labs v. 8.0 program were employed to calculate a set of physicochemical parameters for the investigated compounds. Correlation and multiple linear regression analysis were used to search for the best QSAR equations. The correlations obtained for the compounds studied represent their interactions with the proposed biochromatographic models. The good multivariate relationships (R2 = 0.78–0.84) obtained by means of regression analysis can be used for predicting the quantitative effect of biological activity of different compounds with 5-HT receptor affinity. “Leave-one-out” (LOO) and “leave-N-out” (LNO) cross-validation methods were used to judge the predictive power of final regression equations. PMID:22619602

  10. CTEP: a novel, potent, long-acting, and orally bioavailable metabotropic glutamate receptor 5 inhibitor.

    PubMed

    Lindemann, Lothar; Jaeschke, Georg; Michalon, Aubin; Vieira, Eric; Honer, Michael; Spooren, Will; Porter, Richard; Hartung, Thomas; Kolczewski, Sabine; Büttelmann, Bernd; Flament, Christophe; Diener, Catherine; Fischer, Christophe; Gatti, Silvia; Prinssen, Eric P; Parrott, Neil; Hoffmann, Gerhard; Wettstein, Joseph G

    2011-11-01

    The metabotropic glutamate receptor 5 (mGlu5) is a glutamate-activated class C G protein-coupled receptor widely expressed in the central nervous system and clinically investigated as a drug target for a range of indications, including depression, Parkinson's disease, and fragile X syndrome. Here, we present the novel potent, selective, and orally bioavailable mGlu5 negative allosteric modulator with inverse agonist properties 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP binds mGlu5 with low nanomolar affinity and shows >1000-fold selectivity when tested against 103 targets, including all known mGlu receptors. CTEP penetrates the brain with a brain/plasma ratio of 2.6 and displaces the tracer [(3)H]3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime (ABP688) in vivo in mice from brain regions expressing mGlu5 with an average ED(50) equivalent to a drug concentration of 77.5 ng/g in brain tissue. This novel mGlu5 inhibitor is active in the stress-induced hyperthermia procedure in mice and the Vogel conflict drinking test in rats with minimal effective doses of 0.1 and 0.3 mg/kg, respectively, reflecting a 30- to 100-fold higher in vivo potency compared with 2-methyl-6-(phenylethynyl)pyridine (MPEP) and fenobam. CTEP is the first reported mGlu5 inhibitor with both long half-life of approximately 18 h and high oral bioavailability allowing chronic treatment with continuous receptor blockade with one dose every 48 h in adult and newborn animals. By enabling long-term treatment through a wide age range, CTEP allows the exploration of the full therapeutic potential of mGlu5 inhibitors for indications requiring chronic receptor inhibition. PMID:21849627

  11. Two short-acting kappa opioid receptor antagonists (zyklophin and LY2444296) exhibited different behavioral effects from the long-acting antagonist norbinaltorphimine in mouse anxiety tests.

    PubMed

    Huang, Peng; Yakovleva, Tatyana; Aldrich, Jane V; Tunis, Julia; Parry, Christopher; Liu-Chen, Lee-Yuan

    2016-02-26

    Prototypical long-acting kappa opioid receptor (KOPR) antagonists [e.g., norbinaltorphimine (norBNI)] have been reported to exert anxiolytic-like effects in several commonly used anxiety tests in rodents including the novelty-induced hypophagia (NIH) and elevated plus maze (EPM) tests. It remains unknown if the short-acting KOPR antagonists (e.g., zyklophin and LY2444296) have similar effects. In this study effects of zyklophin and LY2444296 (s.c.) were investigated in the NIH and EPM tests in mice 1h post-injection and compared with norBNI (i.p.) 48h post-administration. In the NIH test, zyklophin at 3 and 1mg/kg, but not 0.3mg/kg, or LY2444296 at 30mg/kg decreased the latency of palatable food consumption in novel cages, but had no effect in training cages, similar to norBNI (10mg/kg). Zyklophin at 3 or 1mg/kg increased or had a trend of increasing the amount of palatable food consumption in novel cages, with no effects in training cages, further indicating its anxiolytic-like effect, but norBNI (10mg/kg) and LY2444296 (30mg/kg) did not. In the EPM test, norBNI (10mg/kg) increased open arm time and % open arm entries or time, but zyklophin at all three doses and LY2444296 (30mg/kg) had no effects. In addition, zyklophin at 3mg/kg increased numbers of close and total arm entries on EPM, suggesting increased activity; however, norBNI and LY2444296 had no effects on close and total arm entries. Thus, all three KOPR antagonists had anxiolytic-like effects in the NIH test. However, only the long-acting one (norBNI), but not the short-acting ones (zyklophin and LY2444296), demonstrated anti-anxiety like effects in the EPM test. It remains to be investigated if the differences are due to the differences in their durations of action and/or pharmacodynamic properties. PMID:26780565

  12. LGR4 acts as a key receptor for R-spondin 2 to promote osteogenesis through Wnt signaling pathway.

    PubMed

    Zhu, Chao; Zheng, Xin-Feng; Yang, Yue-Hua; Li, Bo; Wang, Yu-Ren; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2016-08-01

    R-spondin proteins are identified as secreted agonists of the canonical Wnt/β-catenin signaling pathway, and leucine-rich repeat-containing G-protein-coupled receptors (LGR) are recognized as R-spondin receptors. The potential role of R-spondin 2 (Rspo2) and LGR4 in mediating osteogenesis remains poorly understood. In our in vitro experiments, we found that Rspo2 could promote osteogenesis through activating the Wnt signaling pathway in MC3T3-E1 cells. However, this effect of Rsop2 disappeared in the cells with functional disruption of LGR4. Meanwhile, Rspo2 significantly inhibited osteoclastogenesis and this effect of Rspo2 was dependent on the presence of osteoblasts with normal function of LGR4. In our in vivo experiments, we found that application of exogenous Rspo2 rescued the bone loss and improved the microarchitecture of bone in OVX mice. Rspo2 could be a positive regulator of bone metabolism through activating the canonical Wnt/β-catenin signaling, and LGR4 acted as a key receptor for Rspo2 to promote osteogenesis. PMID:27140682

  13. delta-Opioid receptors are more efficiently coupled to adenylyl cyclase than to L-type Ca(2+) channels in transfected rat pituitary cells.

    PubMed

    Prather, P L; Song, L; Piros, E T; Law, P Y; Hales, T G

    2000-11-01

    Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which delta-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH(3) cells with cDNAs encoding for delta-opioid receptors. In cells transfected with a relatively low delta-receptor density of 0.55 pmol/mg of protein (GH(3)DOR), activation of delta-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca(2+) current. In contrast, activation of delta-receptors in a clone that contained a higher density of delta-receptors (2.45 pmol/mg of protein) and was also coexpressed with mu-opioid receptors (GH(3)MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca(2+) current. The purpose of the present study was to determine whether these observations resulted from differences in delta-opioid receptor density between clones or interaction between delta- and mu-opioid receptors to allow the activation of different G proteins and signaling to Ca(2+) channels. Using the delta-opioid receptor alkylating agent SUPERFIT, reduction of available delta-opioid receptors in GH(3)MORDOR cells to a density similar to that of delta-opioid receptors in the GH(3)DOR clone resulted in abolishment of coupling to Ca(2+) channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by delta-opioid receptors in GH(3)MORDOR cells, delta-opioid receptor activation in GH(3)DOR cells resulted in coupling to the identical pattern of G proteins seen in GH(3)MORDOR cells. These findings suggest that different threshold densities of delta-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that delta-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca(2

  14. Looking for the role of cannabinoid receptor heteromers in striatal function.

    PubMed

    Ferré, Sergi; Goldberg, Steven R; Lluis, Carme; Franco, Rafael

    2009-01-01

    The introduction of two concepts, "local module" and "receptor heteromer", facilitates the understanding of the role of interactions between different neurotransmitters in the brain. In artificial cell systems, cannabinoid CB(1) receptors form receptor heteromers with dopamine D2, adenosine A2A and mu opioid receptors. There is indirect but compelling evidence for the existence of the same CB1 receptor heteromers in striatal local modules centered in the dendritic spines of striatal GABAergic efferent neurons, particularly at a postsynaptic location. Their analysis provides new clues for the role of endocannabinoids in striatal function, which cannot only be considered as retrograde signals that inhibit neurotransmitter release. Recent studies using a new method to detect heteromerization of more than two proteins, which consists of sequential BRET-FRET (SRET) analysis, has demonstrated that CB1, D2 and A2A receptors can form heterotrimers in transfected cells. It is likely that functional CB1-A2A-D2 receptor heteromers can be found where they are highly co-expressed, in the dendritic spines of GABAergic enkephalinergic neurons. The functional properties of these multiple receptor heteromers and their role in striatal function need to be determined. PMID:18691604

  15. Epinephrine and norepinephrine act as potent agonists at the recombinant human dopamine D4 receptor.

    PubMed

    Lanau, F; Zenner, M T; Civelli, O; Hartman, D S

    1997-02-01

    The catecholamines dopamine (DA), epinephrine (EP), and norepinephrine (NE) play important roles in learning and memory, emotional states, and control of voluntary movement, as well as cardiovascular and kidney function. They activate distinct but overlapping neuronal pathways through five distinct DA receptors (D1R-D5R) and at least 10 different adrenergic receptors (alpha 1a/b/c, alpha 2a/b/c-1/c-2, and beta 1/beta 2/beta 3). The D4R, which is localized to mesolimbic areas of the brain implicated in affective and emotional behavior, has a deduced amino acid sequence with homology to both adrenergic and dopaminergic receptor subtypes. We report here that DA, EP, and NE all show binding in the nanomolar range to three isoforms of the recombinant human D4R (hD4R): D4.2, D4.4, and D4.7. Submicromolar concentrations of DA, EP, and NE were sufficient to activate hD4R isoforms in two different functional assays: agonist-induced guanosine 5'-O-(3-[35S]thiotriphosphate) binding and modulation of adenylyl cyclase activity. DA was approximately fivefold more potent than EP and NE at the D4R, whereas activation of the human D2R required at least 100-fold higher catecholamine concentrations. Functional activation of the D4R by multiple neurotransmitters may provide a novel mechanism for integration of catecholamine signaling in the brain and periphery. PMID:9003072

  16. Two FGF Receptor Kinase Molecules Act in Concert to Recruit and Transphosphorylate Phospholipase Cγ.

    PubMed

    Huang, Zhifeng; Marsiglia, William M; Basu Roy, Upal; Rahimi, Nader; Ilghari, Dariush; Wang, Huiyan; Chen, Huaibin; Gai, Weiming; Blais, Steven; Neubert, Thomas A; Mansukhani, Alka; Traaseth, Nathaniel J; Li, Xiaokun; Mohammadi, Moosa

    2016-01-01

    The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cγ (PLCγ), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCγ complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCγ by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation. PMID:26687682

  17. Peripheral Administration of a Long-Acting Peptide Oxytocin Receptor Agonist Inhibits Fear-Induced Freezing.

    PubMed

    Modi, Meera E; Majchrzak, Mark J; Fonseca, Kari R; Doran, Angela; Osgood, Sarah; Vanase-Frawley, Michelle; Feyfant, Eric; McInnes, Heather; Darvari, Ramin; Buhl, Derek L; Kablaoui, Natasha M

    2016-08-01

    Oxytocin (OT) modulates the expression of social and emotional behaviors and consequently has been proposed as a pharmacologic treatment of psychiatric diseases, including autism spectrum disorders and schizophrenia; however, endogenous OT has a short half-life in plasma and poor permeability across the blood-brain barrier. Recent efforts have focused on the development of novel drug delivery methods to enhance brain penetration, but few efforts have aimed at improving its half-life. To explore the behavioral efficacy of an OT analog with enhanced plasma stability, we developed PF-06655075 (PF1), a novel non-brain-penetrant OT receptor agonist with increased selectivity for the OT receptor and significantly increased pharmacokinetic stability. PF-06478939 was generated with only increased stability to disambiguate changes to selectivity versus stability. The efficacy of these compounds in evoking behavioral effects was tested in a conditioned fear paradigm. Both central and peripheral administration of PF1 inhibited freezing in response to a conditioned fear stimulus. Peripheral administration of PF1 resulted in a sustained level of plasma concentrations for greater than 20 hours but no detectable accumulation in brain tissue, suggesting that plasma or cerebrospinal fluid exposure was sufficient to evoke behavioral effects. Behavioral efficacy of peripherally administered OT receptor agonists on conditioned fear response opens the door to potential peripheral mechanisms in other behavioral paradigms, whether they are mediated by direct peripheral activation or feed-forward responses. Compound PF1 is freely available as a tool compound to further explore the role of peripheral OT in behavioral response. PMID:27217590

  18. Peripheral Administration of a Long-Acting Peptide Oxytocin Receptor Agonist Inhibits Fear-Induced Freezing

    PubMed Central

    Modi, Meera E.; Majchrzak, Mark J.; Fonseca, Kari R.; Doran, Angela; Osgood, Sarah; Vanase-Frawley, Michelle; Feyfant, Eric; McInnes, Heather; Darvari, Ramin; Buhl, Derek L.

    2016-01-01

    Oxytocin (OT) modulates the expression of social and emotional behaviors and consequently has been proposed as a pharmacologic treatment of psychiatric diseases, including autism spectrum disorders and schizophrenia; however, endogenous OT has a short half-life in plasma and poor permeability across the blood-brain barrier. Recent efforts have focused on the development of novel drug delivery methods to enhance brain penetration, but few efforts have aimed at improving its half-life. To explore the behavioral efficacy of an OT analog with enhanced plasma stability, we developed PF-06655075 (PF1), a novel non–brain-penetrant OT receptor agonist with increased selectivity for the OT receptor and significantly increased pharmacokinetic stability. PF-06478939 was generated with only increased stability to disambiguate changes to selectivity versus stability. The efficacy of these compounds in evoking behavioral effects was tested in a conditioned fear paradigm. Both central and peripheral administration of PF1 inhibited freezing in response to a conditioned fear stimulus. Peripheral administration of PF1 resulted in a sustained level of plasma concentrations for greater than 20 hours but no detectable accumulation in brain tissue, suggesting that plasma or cerebrospinal fluid exposure was sufficient to evoke behavioral effects. Behavioral efficacy of peripherally administered OT receptor agonists on conditioned fear response opens the door to potential peripheral mechanisms in other behavioral paradigms, whether they are mediated by direct peripheral activation or feed-forward responses. Compound PF1 is freely available as a tool compound to further explore the role of peripheral OT in behavioral response. PMID:27217590

  19. Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

    PubMed

    Wang, Wei-jia; Wang, Yuan; Chen, Hang-zi; Xing, Yong-zhen; Li, Feng-wei; Zhang, Qian; Zhou, Bo; Zhang, Hong-kui; Zhang, Jie; Bian, Xue-li; Li, Li; Liu, Yuan; Zhao, Bi-xing; Chen, Yan; Wu, Rong; Li, An-zhong; Yao, Lu-ming; Chen, Ping; Zhang, Yi; Tian, Xu-yang; Beermann, Friedrich; Wu, Mian; Han, Jiahuai; Huang, Pei-qiang; Lin, Tianwei; Wu, Qiao

    2014-02-01

    Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death. PMID:24316735

  20. C-type lectins do not act as functional receptors for filovirus entry into cells

    SciTech Connect

    Matsuno, Keita; Nakayama, Eri; Noyori, Osamu; Marzi, Andrea; Ebihara, Hideki; Irimura, Tatsuro; Feldmann, Heinz; Takada, Ayato

    2010-12-03

    Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

  1. Adenosine A(3) receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Vacek, Antonín; Streitová, Denisa

    2007-07-01

    The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A(3) receptor agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IB-MECA given at single doses of 200nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions. PMID:17383145

  2. Differential desensitization of mu- and delta- opioid receptors in selected neural pathways following chronic morphine treatment.

    PubMed Central

    Noble, F.; Cox, B. M.

    1996-01-01

    -mediated inhibition without modification of mu-opioid receptor-mediated inhibition was observed. An indirect mechanism probably involving dopaminergic systems is proposed to explain the desensitization of delta-mediated responses and the lack of mu-opioid receptor desensitization after chronic morphine treatment in caudate putamen and nucleus accumbens. 7. These results suggest that adaptive responses occurring during chronic morphine administration are not identical in all opiate-sensitive neural populations. PMID:8825358

  3. Options for intensification of basal insulin in type 2 diabetes: Premeal insulin or short-acting GLP-1 receptor agonists?

    PubMed

    Darmon, P; Raccah, D

    2015-12-01

    Type 2 diabetes is an evolutive disease with a progressive defect of beta-cell insulin secretion. This characteristic points to a need for treatment that takes into account such a natural history. When oral antidiabetic drugs fail to achieve the patient's target HbA1c level, basal insulin treatment is usually initiated and titrated in association with oral drugs to manage fasting hyperglycaemia. Over a period of time, it is enough to simply achieve the HbA1c target. However, when even a good fasting blood glucose level is no longer sufficient to control overall glycaemia, then prandial treatment must be combined with the titrated basal insulin to deal with the postprandial hyperglycaemia responsible for the elevation of HbA1c. Of the different therapeutic options now available for this, rapid-acting insulins and GLP-1 receptor agonists (RAs) can be used. Rapid-acting insulins can be added either at each meal, achieving full insulin supplementation with a basal-bolus regimen, or at the main meal only as a "basal-plus" regimen. Compared with the full basal-bolus, the basal-plus strategy is associated with fewer injections, yet provides similar efficacy in terms of HbA1c improvement, but with less weight gain and lower hypoglycaemic risk. As for GLP-1 RAs, numerous studies, and especially those using short-acting GLP-1 RAs, have demonstrated more pronounced effects on postprandial hyperglycaemia, good complementary effects with basal insulin, and significant improvement of HbA1c with no weight gain and a low risk of hypoglycaemia. Similarly, direct and indirect comparisons of the use of rapid-acting insulins and GLP-1 RAs to intensify basal insulin have shown comparable efficacy in terms of HbA1c control, but with less weight gain and fewer hypoglycaemic episodes with GLP-1 RAs. PMID:26774016

  4. CEACAM6 acts as a receptor for adherent-invasive E. coli, supporting ileal mucosa colonization in Crohn disease.

    PubMed

    Barnich, Nicolas; Carvalho, Frédéric A; Glasser, Anne-Lise; Darcha, Claude; Jantscheff, Peter; Allez, Matthieu; Peeters, Harald; Bommelaer, Gilles; Desreumaux, Pierre; Colombel, Jean-Frédéric; Darfeuille-Michaud, Arlette

    2007-06-01

    The ileal mucosa of Crohn disease (CD) patients is abnormally colonized by adherent-invasive E. coli (AIEC) that are able to adhere to and invade intestinal epithelial cells. Here, we show that CD-associated AIEC strains adhere to the brush border of primary ileal enterocytes isolated from CD patients but not controls without inflammatory bowel disease. AIEC adhesion is dependent on type 1 pili expression on the bacterial surface and on carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression on the apical surface of ileal epithelial cells. We report also that CEACAM6 acts as a receptor for AIEC adhesion and is abnormally expressed by ileal epithelial cells in CD patients. In addition, our in vitro studies show that there is increased CEACAM6 expression in cultured intestinal epithelial cells after IFN-gamma or TNF-alpha stimulation and after infection with AIEC bacteria, indicating that AIEC can promote its own colonization in CD patients. PMID:17525800

  5. Receptor tyrosine phosphatase CLR-1 acts in skin cells to promote sensory dendrite outgrowth.

    PubMed

    Liu, Xianzhuang; Wang, Xiangming; Shen, Kang

    2016-05-01

    Sensory dendrite morphogenesis is directed by intrinsic and extrinsic factors. The extracellular environment plays instructive roles in patterning dendrite growth and branching. However, the molecular mechanism is not well understood. In Caenorhabditis elegans, the proprioceptive neuron PVD forms highly branched sensory dendrites adjacent to the hypodermis. We report that receptor tyrosine phosphatase CLR-1 functions in the hypodermis to pattern the PVD dendritic branches. Mutations in clr-1 lead to loss of quaternary branches, reduced secondary branches and increased ectopic branches. CLR-1 is necessary for the dendrite extension but not for the initial filopodia formation. Its role is dependent on the intracellular phosphatase domain but not the extracellular adhesion domain, indicating that it functions through dephosphorylating downstream factors but not through direct adhesion with neurons. Genetic analysis reveals that clr-1 also functions in parallel with SAX-7/DMA-1 pathway to control PVD primary dendrite development. We provide evidence of a new environmental factor for PVD dendrite morphogenesis. PMID:26968353

  6. Differential antagonism of tetramethylenedisulfotetramine-induced seizures by agents acting at NMDA and GABAA receptors

    PubMed Central

    Shakarjian, Michael P.; Velíšková, Jana; Stanton, Patric K.; Velíšek, Libor

    2012-01-01

    Tetramethylenedisulfotetramine (TMDT) is a highly lethal neuroactive rodenticide responsible for many accidental and intentional poisonings in mainland China. Ease of synthesis, water solubility, potency, and difficulty to treat make TMDT a potential weapon for terrorist activity. We characterized TMDT-induced convulsions and mortality in male C57BL/6 mice. TMDT (ip) produced a continuum of twitches, clonic, and tonic-clonic seizures decreasing in onset latency and increasing in severity with increasing dose; 0.4 mg/kg was 100% lethal. The NMDA antagonist, ketamine (35 mg/kg) injected ip immediately after the first TMDT-induced seizure, did not change number of tonic-clonic seizures or lethality, but increased the number of clonic seizures. Doubling the ketamine dose decreased tonic-clonic seizures and eliminated lethality through a 60 min observation period. Treating mice with another NMDA antagonist, MK-801, 0.5 or 1 mg/kg ip, showed similar effects as low and high doses of ketamine, respectively, and prevented lethality, converting status epilepticus EEG activity to isolated interictal discharges. Treatment with these agents 15 min prior to TMDT administration did not increase their effectiveness. Post-treatment with the GABAA receptor allosteric enhancer diazepam (5 mg/kg) greatly reduced seizure manifestations and prevented lethality 60 min post-TMDT, but ictal events were evident in EEG recordings and, hours post-treatment, mice experienced status epilepticus and died. Thus, TMDT is a highly potent and lethal convulsant for which single-dose benzodiazepine treatment is inadequate in managing electrographic seizures or lethality. Repeated benzodiazepine dosing or combined application of benzodiazepines and NMDA receptor antagonists are more likely to be effective in treating TMDT poisoning. PMID:23022509

  7. Identification of naphthoylindoles acting on cannabinoid receptors based on their fragmentation patterns under ESI-QTOFMS.

    PubMed

    Sekuła, Karolina; Zuba, Dariusz; Stanaszek, Roman

    2012-05-01

    'Herbal highs' have been advertised as legal and natural substitutes to cannabis, but a detailed examination of these products has revealed that the herbal matrix is laced with synthetic substances that mimic the effects of marijuana. Producers select the ingredients based on the results of scientific studies on the affinities of different chemicals to cannabinoid receptors. Naphthoylindoles have turned out to be the most popular class of substances identified in the products. Legal actions taken in order to tackle the problem of uncontrolled access to one substance have usually resulted in the marketing of derivatives or analogues. In the study, the mass spectral behavior of twelve synthetic cannabinoids from the naphthoylindole family under electrospray ionization (ESI) was investigated. LC-QTOFMS experiments were performed in three modes (low fragmentor voltage, high fragmentor voltage with/without collision energy), and they enabled the identification of protonated molecules and main ions. A general fragmentation pattern under this ionization method was proposed, and mechanisms of ion formation were discussed. The developed procedure allowed the determination of substituent groups of the core naphthoylindole structure and distinction between positional isomers. The obtained results were used for the prediction of the ESI-MS spectra for many naphthoylindoles with a high affinity to cannabinoid receptors. Similarities and differences between ESI-MS and electron impact-MS spectra of naphthoylindoles were discussed. The developed identification process was presented on an example of an analysis of an unknown herbal material, in which JWH-007 was finally identified. Knowledge of the fragmentation mechanisms of naphthoylindoles could also be used by other researchers for identification of unknown substances in this chemical family. PMID:22576877

  8. The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies

    PubMed Central

    Freeman, Brian C.; Felts, Sara J.; Toft, David O.; Yamamoto, Keith R.

    2000-01-01

    Multiple molecular chaperones, including Hsp90 and p23, interact with members of the intracellular receptor (IR) family. To investigate p23 function, we compared the effects of three p23 proteins on IR activities, yeast p23 (sba1p) and the two human p23 homologs, p23 and tsp23. We found that Sba1p was indistinguishable from human p23 in assays of seven IR activities in both animal cells and in yeast; in contrast, certain effects of tsp23 were specific to that homolog. Transcriptional activation by two IRs was increased by expression of any of the p23 species, whereas activation by five other IRs was decreased by Sba1p or p23, and unaffected by tsp23. p23 was expressed in all tissues examined except striated and cardiac muscle, whereas tsp23 accumulated in a complementary pattern; hence, p23 proteins might contribute to tissue-specific differences in IR activities. Unlike Hsp90, which acts on IR aporeceptors to stimulate ligand potency (i.e., hormone-binding affinity), p23 proteins acted on IR holoreceptors to alter ligand efficiencies (i.e., transcriptional activation activity). Moreover, the p23 effects developed slowly, requiring prolonged exposure to hormone. In vitro, p23 interacted preferentially with hormone–receptor–response element ternary complexes, and stimulated receptor–DNA dissociation. The dissociation was reversed by addition of a fragment of the GRIP1 coactivator, suggesting that the two reactions may be in competition in vivo. Our findings suggest that p23 functions at one or more late steps in IR-mediated signal transduction, perhaps including receptor recycling and/or reversal of the response. PMID:10691735

  9. Pharmacokinetics and pharmacodynamics of LCZ696, a novel dual-acting angiotensin receptor-neprilysin inhibitor (ARNi).

    PubMed

    Gu, Jessie; Noe, Adele; Chandra, Priya; Al-Fayoumi, Suliman; Ligueros-Saylan, Monica; Sarangapani, Ramesh; Maahs, Suzanne; Ksander, Gary; Rigel, Dean F; Jeng, Arco Y; Lin, Tsu-Han; Zheng, Weiyi; Dole, William P

    2010-04-01

    Angiotensin receptor blockade and neprilysin (NEP) inhibition together offer potential benefits for the treatment of hypertension and heart failure. LCZ696 is a novel single molecule comprising molecular moieties of valsartan and NEP inhibitor prodrug AHU377 (1:1 ratio). Oral administration of LCZ696 caused dose-dependent increases in atrial natriuretic peptide immunoreactivity (due to NEP inhibition) in Sprague-Dawley rats and provided sustained, dose-dependent blood pressure reductions in hypertensive double-transgenic rats. In healthy participants, a randomized, double-blind, placebo-controlled study (n = 80) of single-dose (200-1200 mg) and multiple-dose (50-900 mg once daily for 14 days) oral administration of LCZ696 showed that peak plasma concentrations were reached rapidly for valsartan (1.6-4.9 hours), AHU377 (0.5-1.1 hours), and its active moiety, LBQ657 (1.8-3.5 hours). LCZ696 treatment was associated with increases in plasma cGMP, renin concentration and activity, and angiotensin II, providing evidence for NEP inhibition and angiotensin receptor blockade. In a randomized, open-label crossover study in healthy participants (n = 56), oral LCZ696 400 mg and valsartan 320 mg were shown to provide similar exposure to valsartan (geometric mean ratio [90% confidence interval]: AUC(0-infinity) 0.90 [0.82-0.99]). LCZ696 was safe and well tolerated. These data support further clinical development of LCZ696, a novel, orally bioavailable, dual-acting angiotensin receptor-NEP inhibitor (ARNi) for hypertension and heart failure. PMID:19934029

  10. Neuromedin U directly stimulates growth of cultured rat calvarial osteoblast-like cells acting via the NMU receptor 2 isoform.

    PubMed

    Rucinski, Marcin; Ziolkowska, Agnieszka; Tyczewska, Marianna; Szyszka, Marta; Malendowicz, Ludwik K

    2008-09-01

    The neuromedin U (NMU) system is composed of NMU, neuromedin S (NMS) and their receptors NMUR1 and NMUR2. This system is involved in the regulation of energy homeostasis, neuroendocrine functions, immune response, circadian rhythm and spermatogenesis. The present study aimed to investigate the possible role of the NMU system in regulating functions of cultured rat calvarial osteoblast-like (ROB) cells. By using QPCR, high expression of NMU mRNA was found in freshly isolated ROB cells while after 7, 14, and 21 days of culture, expression of the studied gene was very low. In contrast, NMUR2 mRNA expression in freshly isolated ROB cells was negligible and very high in cultured cells. The highest NMUR2 mRNA expression was observed at day 7, and was followed by lower levels at days 14 and 21 of culture. Neither NMS nor NMUR1 mRNA was found in studied cells. Exposure of cultured ROB cells to NMU8 at concentrations 10(-6) to 10(-10) M had no effect on expression levels of the genes. During the entire culture period, NMU8 did not affect osteocalcin production, but stimulated proliferative activity of ROB cells at days 14 and 21 of culture. Thus, we demonstrated that cultured rat calvarial osteoblast-like cells are provided with NMUR2, the receptor isoform typical for the central nervous system. Acting via this receptor NMU8 stimulates proliferation of cultured cells and has no effect on their differentiated function (osteocalcin secretion). PMID:18698496

  11. 2-Guanidine-4-methylquinazoline acts as a novel competitive antagonist of A type γ-aminobutyric acid receptors.

    PubMed

    Xiao, Xian; Zhu, Michael X; Xu, Tian-Le

    2013-12-01

    The pentameric A type γ-aminobutyric acid receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the nervous system and have long been considered as important pharmaceutical targets for the treatment of multiple neurological or psychological disorders. Here, we show that 2-guanidine-4-methylquinazoline (GMQ), a recently identified acid-sensing ion channel (ASIC) modulator, strongly and preferentially inhibits GABAAR among the major neurotransmitter-gated ion channels in cultured rat hippocampal neurons. GMQ inhibited GABA (1 μM)-induced currents in a competitive manner, with an IC50 (0.39±0.05 μM) comparable to that of bicuculline. Schild analysis revealed a slope of 1.04±0.06 for GMQ on α1β2 GABAARs expressed in HEK293T cells. Single-channel analysis showed that GMQ decreased open probability of GABAARs without affecting conductance. Moreover, GMQ inhibited GABAergic neurotransmission in hippocampal neurons, while having no significant effect on the basal field excitatory postsynaptic potentials (fEPSPs) and the intrinsic excitability of neurons. Using site-directed mutagenesis, we further demonstrated that mutations at Glu155 of β2 subunit and Phe64 of α1 subunit, both located inside the GABA binding pocket, profoundly decreased the sensitivity of the receptor to both GABA and GMQ. Interestingly, these mutations did not significantly affect the inhibition by amiloride, a diuretic structurally similar to GMQ and a known GABAAR inhibitor. We conclude that GMQ represents a novel chemical structure that acts, possibly, by competing with GABA binding to GABAARs. It is anticipated that GMQ and its analogs will facilitate the development of new chemical probes for GABAARs. PMID:23916476

  12. 2-[4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy]-N-(methylsulfonyl)acetamide (NS-304), an orally available and long-acting prostacyclin receptor agonist prodrug.

    PubMed

    Kuwano, Keiichi; Hashino, Asami; Asaki, Tetsuo; Hamamoto, Taisuke; Yamada, Tetsuhiro; Okubo, Kaori; Kuwabara, Kenji

    2007-09-01

    Prostacyclin (PGI(2)) and its analogs are useful for the treatment of various vascular disorders, but their half-lives are too short for widespread clinical application. To overcome this drawback, we have synthesized a novel diphenylpyrazine derivative, 2-[4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy]-N-(methylsulfonyl)acetamide (NS-304), a prodrug of the active form [4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy]acetic acid (MRE-269). NS-304 is an orally available and potent agonist for the PGI(2) receptor (IP receptor). The inhibition constant (K(i)) of MRE-269 for the human IP receptor was 20 nM; in contrast, the K(i) values for other prostanoid receptors were >2.6 microM. MRE-269 was therefore a highly selective agonist for the IP receptor. The plasma concentrations of MRE-269 remained near peak levels for more than 8 h after oral administration of NS-304 to rats and dogs, and NS-304 increased femoral skin blood flow in rats in a long-lasting manner without affecting the hemodynamics. These findings indicate that NS-304 acts as a long-acting IP receptor agonist in vivo. The continuous vasodilation evoked by NS-304 was not attenuated by repeated treatment, indicating that NS-304 is unlikely to cause severe desensitization of the IP receptor in rats. Moreover, a microdose pharmacokinetic study in which NS-304 was orally administered to healthy male volunteers showed conversion of NS-304 to MRE-269 and a long plasma elimination half-life for MRE-269 (7.9 h). In conclusion, NS-304 is an orally available and long-acting IP receptor agonist prodrug, and its active form, MRE-269, is highly selective for the IP receptor. Therefore, NS-304 is a promising drug candidate for various vascular diseases, especially pulmonary arterial hypertension and arteriosclerosis obliterans. PMID:17545310

  13. Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin.

    PubMed Central

    Nyberg, G; Strömberg, N; Jonsson, A; Karlsson, K A; Normark, S

    1990-01-01

    Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was

  14. Differential receptor binding characteristics of consecutive phenylalanines in micro-opioid specific peptide ligand endomorphin-2.

    PubMed

    Honda, Takeshi; Shirasu, Naoto; Isozaki, Kaname; Kawano, Michiaki; Shigehiro, Daiki; Chuman, Yoshiro; Fujita, Tsugumi; Nose, Takeru; Shimohigashi, Yasuyuki

    2007-06-01

    Endogenous opioid peptides consist of a conserved amino acid residue of Phe(3) and Phe(4), although their binding modes for opioid receptors have not been elucidated in detail. Endomorphin-2, which is highly selective and specific for the mu opioid receptor, possesses two Phe residues at the consecutive positions 3 and 4. In order to clarify the role of Phe(3) and Phe(4) in binding to the mu receptor, we synthesized a series of analogs in which Phe(3) and Phe(4) were replaced by various amino acids. It was found that the aromaticity of the Phe-beta-phenyl groups of Phe(3) and Phe(4) is a principal determinant of how strongly it binds to the receptor, although better molecular hydrophobicity reinforces the activity. The receptor binding subsites of Phe(3) and Phe(4) of endomorphin-2 were found to exhibit different structural requirements. The results suggest that [Trp(3)]endomorphin-2 (native endomorphin-1) and endomorphin-2 bind to different receptor subclasses. PMID:17395470

  15. Endocytosis as a Biological Response in Receptor Pharmacology: Evaluation by Fluorescence Microscopy

    PubMed Central

    Varela, María J.; de la Rocha, Arlet M. Acanda; Fernandez-Troyano, Juan C.; Barreiro, R. Belén; Lopez-Gimenez, Juan F.

    2015-01-01

    The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method –the Q-Endosomes algorithm– that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution. PMID:25849355

  16. GABAA receptor-acting neurosteroids: A role in the development and regulation of the stress response

    PubMed Central

    Gunn, Benjamin G.; Cunningham, Linda; Mitchell, Scott G.; Swinny, Jerome D.; Lambert, Jeremy J.; Belelli, Delia

    2015-01-01

    Regulation of hypothalamic–pituitary–adrenocortical (HPA) axis activity by stress is a fundamental survival mechanism and HPA-dysfunction is implicated in psychiatric disorders. Adverse early life experiences, e.g. poor maternal care, negatively influence brain development and programs an abnormal stress response by encoding long-lasting molecular changes, which may extend to the next generation. How HPA-dysfunction leads to the development of affective disorders is complex, but may involve GABAA receptors (GABAARs), as they curtail stress-induced HPA axis activation. Of particular interest are endogenous neurosteroids that potently modulate the function of GABAARs and exhibit stress-protective properties. Importantly, neurosteroid levels rise rapidly during acute stress, are perturbed in chronic stress and are implicated in the behavioural changes associated with early-life adversity. We will appraise how GABAAR-active neurosteroids may impact on HPA axis development and the orchestration of the stress-evoked response. The significance of these actions will be discussed in the context of stress-associated mood disorders. PMID:24929099

  17. Ethanol acts directly on extrasynaptic subtypes of GABAA receptors to increase tonic inhibition

    PubMed Central

    Santhakumar, Vijayalakshmi; Wallner, Martin; Otis, Thomas S.

    2007-01-01

    Based on the similarity of ethanol intoxication to the behavioral effects of drugs known to target GABAA receptors (GABARs) it has been suspected for decades that ethanol facilitates the activity of GABA. Even so, it has been surprisingly difficult to identify molecular targets of ethanol. Research conducted over the past several years suggests that a subclass of GABARs (those containing δ subunits) responds in a relevant concentration range to ethanol. Although δ subunit-containing GABARs are not ubiquitously expressed at inhibitory synapses like their γ subunit-containing, synaptic counterparts, they are found in many neurons in extrasynaptic locations. Here they give rise to a tonic form of inhibition that can potently suppress neuronal excitability. Studies have shown that both recombinant and native δ subunit-containing GABARs: 1) are modulated by behaviorally-relevant (i.e. low millimolar) concentrations of ethanol, 2) directly bind ethanol over the same concentration range, 3) show altered function upon single amino substitutions linked to changes in behavioral responsiveness to ethanol, and 4) are a site of action of Ro15-4513, a competitive antagonist of ethanol binding and a drug which prevents many of the behavioral aspects of ethanol intoxication. Despite such comprehensive evidence, however, the field is not free from controversy. This review evaluates published data for and against a central role of δ subunit-containing GABARs in ethanol actions and suggests future directions that might help settle points of controversy. PMID:17591544

  18. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans

    PubMed Central

    Wang, Jiali; Liu, Chengzhi; Lu, Huizhi; Liu, Mengjia; Zhao, Ye; Tian, Bing; Wang, Liangyan; Hua, Yuejin

    2016-01-01

    The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2) treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998), Lon proteases (dr0349 and dr1974), NADH-quinone oxidoreductase (dr1506), thiosulfate sulfurtransferase (dr2531), the DNA repair protein UvsE (dr1819), PprA (dra0346), and RecN (dr1447), are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways. PMID:27182600

  19. THE FUNGICIDE PROCYMIDONE ALTERS SEXUAL DIFFERENTIATION IN THE MALE RAT BY ACTING AS AN ANDROGEN-RECEPTOR ANTAGONIST IN VIVO AND IN VITRO

    EPA Science Inventory

    The fungicide procymidone alters sexual differentiation in the male rat by acting as an androgen-receptor antagonist in vivo and in vitro.

    Ostby J, Kelce WR, Lambright C, Wolf CJ, Mann P, Gray LE Jr.

    Endocrinology Branch, National Health and Environmental Effects Re...

  20. Structures of an ActRIIB:activin A complex reveal a novel binding mode for TGF-beta ligand:receptor interactions

    SciTech Connect

    Thompson, T.B.; Woodruff, T.K.; Jardetzky, T.S.

    2010-03-08

    The TGF-{beta} superfamily of ligands and receptors stimulate cellular events in diverse processes ranging from cell fate specification in development to immune suppression. Activins define a major subgroup of TGF-{beta} ligands that regulate cellular differentiation, proliferation, activation and apoptosis. Activins signal through complexes formed with type I and type II serine/threonine kinase receptors. We have solved the crystal structure of activin A bound to the extracellular domain of a type II receptor, ActRIIB, revealing the details of this interaction. ActRIIB binds to the outer edges of the activin finger regions, with the two receptors juxtaposed in close proximity, in a mode that differs from TGF-{beta}3 binding to type II receptors. The dimeric activin A structure differs from other known TGF-{beta} ligand structures, adopting a compact folded-back conformation. The crystal structure of the complex is consistent with recruitment of two type I receptors into a close packed arrangement at the cell surface and suggests that diversity in the conformational arrangements of TGF-{beta} ligand dimers could influence cellular signaling processes.

  1. CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo.

    PubMed

    Rossi, Mario; Ruiz de Azua, Inigo; Barella, Luiz F; Sakamoto, Wataru; Zhu, Lu; Cui, Yinghong; Lu, Huiyan; Rebholz, Heike; Matschinsky, Franz M; Doliba, Nicolai M; Butcher, Adrian J; Tobin, Andrew B; Wess, Jürgen

    2015-12-01

    G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic β-cells. β-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of β-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic β-cells, knockdown of CK2α expression, or genetic deletion of CK2α in β-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of β-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on β-cell GPCRs may represent novel therapeutic targets. PMID:26598688

  2. RanBPM Protein Acts as a Negative Regulator of BLT2 Receptor to Attenuate BLT2-mediated Cell Motility*

    PubMed Central

    Wei, Jun-Dong; Kim, Joo-Young; Kim, Ae-Kyoung; Jang, Sung Key; Kim, Jae-Hong

    2013-01-01

    BLT2, a low affinity receptor for leukotriene B4 (LTB4), is a member of the G protein-coupled receptor family and is involved in many signal transduction pathways associated with various cellular phenotypes, including chemotactic motility. However, the regulatory mechanism for BLT2 has not yet been demonstrated. To understand the regulatory mechanism of BLT2, we screened and identified the proteins that bind to BLT2. Using a yeast two-hybrid assay with the BLT2 C-terminal domain as bait, we found that RanBPM, a previously proposed scaffold protein, interacts with BLT2. We demonstrated the specific interaction between BLT2 and RanBPM by GST pulldown assay and co-immunoprecipitation assay. To elucidate the biological function of the RanBPM-BLT2 interaction, we evaluated the effects of RanBPM overexpression or knockdown. We found that BLT2-mediated motility was severely attenuated by RanBPM overexpression and that knockdown of endogenous RanBPM by shRNA strongly promoted BLT2-mediated motility, suggesting a negative regulatory function of RanBPM toward BLT2. Furthermore, we observed that the addition of BLT2 ligands caused the dissociation of BLT2 and RanBPM, thus releasing the negative regulatory effect of RanBPM. Finally, we propose that Akt-induced BLT2 phosphorylation at residue Thr355, which occurs after the addition of BLT2 ligands, is a potential mechanism by which BLT2 dissociates from RanBPM, resulting in stimulation of BLT2 signaling. Taken together, our results suggest that RanBPM acts as a negative regulator of BLT2 signaling to attenuate BLT2-mediated cell motility. PMID:23928309

  3. Facilitation of fear extinction by novelty depends on dopamine acting on D1-subtype dopamine receptors in hippocampus

    PubMed Central

    Menezes, Jefferson; Alves, Niége; Borges, Sidnei; Roehrs, Rafael; de Carvalho Myskiw, Jociane; Furini, Cristiane Regina Guerino; Izquierdo, Ivan; Mello-Carpes, Pâmela B.

    2015-01-01

    Extinction is the learned inhibition of retrieval. Recently it was shown that a brief exposure to a novel environment enhances the extinction of contextual fear in rats, an effect explainable by a synaptic tagging-and-capture process. Here we examine whether this also happens with the extinction of another fear-motivated task, inhibitory avoidance (IA), and whether it depends on dopamine acting on D1 or D5 receptors. Rats were trained first in IA and then in extinction of this task. The retention of extinction was measured 24 h later. A 5-min exposure to a novel environment 30 min before extinction training enhanced its retention. Right after exposure to the novelty, animals were given bilateral intrahippocampal infusions of vehicle (VEH), of the protein synthesis inhibitor anisomycin, of the D1/D5 dopaminergic antagonist SCH23390, of the PKA inhibitor Rp-cAMP or of the PKC inhibitor Gö6976, and of the PKA stimulator Sp-cAMP or of the PKC stimulator PMA. The novelty increased hippocampal dopamine levels and facilitated the extinction, which was inhibited by intrahippocampal protein synthesis inhibitor anisomysin, D1/D5 dopaminerdic antagonist SCH23390, or PKA inhibitor Rp-cAMP and unaffected by PKC inhibitor Gö6976; additionally, the hippocampal infusion of PKA stimulator Sp-cAMP reverts the effect of D1/D5 dopaminergic antagonist SCH 23390, but the infusion of PKC stimulator PMA does not. The results attest to the generality of the novelty effect on fear extinction, suggest that it relies on synaptic tagging and capture, and show that it depends on hippocampal dopamine D1 but not D5 receptors. PMID:25775606

  4. Abscisic Acid Acts as a Blocker of the Bitter Taste G Protein-Coupled Receptor T2R4.

    PubMed

    Pydi, Sai P; Jaggupilli, Appalaraju; Nelson, Ken M; Abrams, Suzanne R; Bhullar, Rajinder P; Loewen, Michele C; Chelikani, Prashen

    2015-04-28

    Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. In humans, 25 T2Rs mediate bitter taste sensation. In addition to the oral cavity, T2Rs are expressed in many extraoral tissues, including the central nervous system, respiratory system, and reproductive system. To understand the mechanistic roles of the T2Rs in oral and extraoral tissues, novel blockers or antagonists are urgently needed. Recently, we elucidated the binding pocket of T2R4 for its agonist quinine, and an antagonist and inhibitory neurotransmitter, γ-aminobutyric acid. This structure-function information about T2R4 led us to screen the plant hormone abscisic acid (ABA), its precursor (xanthoxin), and catabolite phaseic acid for their ability to bind and activate or inhibit T2R4. Molecular docking studies followed by functional assays involving calcium imaging confirmed that ABA is an antagonist with an IC50 value of 34.4 ± 1.1 μM. However, ABA precursor xanthoxin acts as an agonist on T2R4. Interestingly, molecular model-guided site-directed mutagenesis suggests that the T2R4 residues involved in quinine binding are also predominantly involved in binding to the novel antagonist, ABA. The antagonist ability of ABA was tested using another T2R4 agonist, yohimbine. Our results suggest that ABA does not inhibit yohimbine-induced T2R4 activity. The discovery of natural bitter blockers has immense nutraceutical and physiological significance and will help in dissecting the T2R molecular pathways in various tissues. PMID:25844797

  5. A novel, long-acting glucagon-like peptide receptor-agonist: dulaglutide

    PubMed Central

    Gurung, Tara; Shyangdan, Deepson S; O’Hare, Joseph Paul; Waugh, Norman

    2015-01-01

    Background Dulaglutide is a new, long-acting glucagon-like peptide analogue in the treatment of type 2 diabetes. It is available in two doses, 0.75 and 1.5 mg, given by injection once weekly. This systematic review reports the effectiveness and safety of dulaglutide in type 2 diabetes in dual and triple therapy. Methods MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, and conference abstracts were searched from 2005 to August 2014, and updated in January 2015. Company websites and references of included studies were checked for potentially relevant studies. European Medicines Agency and US Food and Drug Administration websites were searched. Results Four trials were included. All were manufacturer-funded randomized controlled trials from the Assessment of Weekly Administration of Dulaglutide in Diabetes (AWARD) program. AWARD-1 compared dulaglutide 1.5 mg against exenatide 10 µg twice daily and placebo, AWARD-2 compared dulaglutide 0.75 and 1.5 mg against insulin glargine, AWARD-5 compared dulaglutide 0.75 and 1.5 mg against sitagliptin 100 mg and placebo, and AWARD-6 compared dulaglutide 1.5 mg against liraglutide 1.8 mg. The duration of follow-up in the trials ranged from 26 to 104 weeks. The primary outcome of all the included trials was change in HbA1c. At 26 weeks, greater HbA1c reductions were seen with dulaglutide than with twice daily exenatide (dulaglutide 1.5/0.75 mg: −1.5%/−1.3%; exe: 0.99%) and sitagliptin (1.5/0.75 mg −1.22%/−1.01%; sitagliptin: −0.6%). HbA1c change was greater with dulaglutide 1.5 mg (−1.08%) than with glargine (−0.63%), but not with dulaglutide 0.75 mg (−0.76%). Dulaglutide 1.5 mg was found to be noninferior to liraglutide 1.8 mg. More patients treated with dulaglutide achieved HbA1c targets of <7% and ≤6.5%. Reduction in weight was greater with dulaglutide than with sitagliptin and exenatide. Hypoglycemia was infrequent. The main adverse events were nausea, diarrhea, and vomiting. Conclusion

  6. PA1 protein, a new competitive decelerator acting at more than one step to impede glucocorticoid receptor-mediated transactivation.

    PubMed

    Zhang, Zhenhuan; Sun, Yunguang; Cho, Young-Wook; Chow, Carson C; Simons, S Stoney

    2013-01-01

    Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (A(max)), the potency of agonists (EC(50)), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses A(max), increases the EC(50), and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC(50), and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene. PMID:23161582

  7. Anxiogenic effects of CGRP within the BNST may be mediated by CRF acting at BNST CRFR1 receptors

    PubMed Central

    Sink, KS; Chung, A; Ressler, KJ; Davis, M; Walker, DL

    2013-01-01

    Calcitonin gene-related peptide (CGRP) acting within the bed nucleus of the stria terminalis (BNST) increases anxiety as well as neural activation in anxiety-related structures, and mediates behavioral stress responses. Similar effects have been described following intra-ventricular as well as intra-BNST infusions of the stress-responsive neuropeptide, corticotropin releasing factor (CRF). Interestingly, CGRP-positive terminals within the lateral division of the BNST form perisomatic baskets around neurons that express CRF, suggesting that BNST CGRP could exert its anxiogenic effects by increasing release of CRF from these neurons. With this in mind, the present set of experiments was designed to examine the role of CRFR1 signaling in the anxiogenic effects of CGRP within the BNST and to determine whether CRF from BNST neurons contributes to these effects. Consistent with previous studies, we found that 400 ng CGRP infused bilaterally into the BNST increased the acoustic startle response and induced anxiety-like behavior in the elevated plus maze compared to vehicle. Both of these effects were attenuated by 10 mg/kg PO of the CRFR1 antagonist, GSK876008. GSK876008 alone did not affect startle. An intra-BNST infusion of the CRFR1 antagonist CP376395 (2 μg) also blocked increases in acoustic startle induced by intra-BNST infusion of CGRP, as did virally-mediated siRNA knockdown of CRF expression locally within the BNST. Together, these results suggest that the anxiogenic effects of intra-BNST CGRP may be mediated by CRF from BNST neurons acting at local CRFR1 receptors. PMID:23376701

  8. Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling

    PubMed Central

    Scearce-Levie, Kimberly; Lieberman, Michael D; Elliott, Heather H; Conklin, Bruce R

    2005-01-01

    Background The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of Gi-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization. Results Removal of phosphorylation sites in the C-terminus of the RASSL resulted in a mutant that was resistant to internalization and less prone to desensitization. Replacement of the C-terminus of the RASSL with the corresponding portion of the mu opioid receptor eliminated the induction of AC superactivation, without disrupting agonist-induced desensitization or internalization. Surprisingly, removal of phosphorylation sites from this chimera resulted in a receptor that is constitutively internalized, even in the absence of agonist. However, the receptor still signals and desensitizes in response to agonist, indicating normal G-protein coupling and partial membrane expression. Conclusions These studies reveal that internalization, desensitization and adenylyl cyclase superactivation, all processes that decrease chronic Gi-receptor signals, are independently regulated. Furthermore, specific mutations can radically alter superactivation or internalization without affecting the efficacy of acute Gi signaling. These mutant RASSLs will be useful for further elucidating the temporal dynamics of the signaling of G protein-coupled receptors in vitro and in vivo. PMID:15707483

  9. Central delta-opioid receptor interactions and the inhibition of reflex urinary bladder contractions in the rat.

    PubMed

    Dray, A; Nunan, L; Wire, W

    1985-07-01

    The in vivo effects of a number of opioid agonists and antagonists were studied on the spontaneous reflex contractions of the urinary bladder recorded isometrically in the rat anesthetized with urethane. All substances were administered into the central nervous system by the intracereboventricular (i.c.v.) or spinal intrathecal (i.t.) route. The conformationally restricted enkephalin analogues [2-D-penicillamine, 5-L-cysteine] enkephalin (DPLCE), [2-D-penicillamine, 5-L-penicillamine] enkephalin (DPLPE) and [2-D-penicillamine, 5-D-penicillamine] enkephalin (DPDPE) produced dose-related inhibition of reflex bladder contractions when administered by the i.c.v. or i.t. route. Both the novel delta-opioid receptor antagonist ICI 154,129 (200-600 micrograms) [N,N-bisallyl-Tyr-Gly-Gly-Psi-(CH2S)-Phe-Leu-OH) and ICI 174,864 (1-3 micrograms) [N,N-dially-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid] attenuated or abolished the effects of DPLCE, DPLPE and DPDPE when administered by the i.c.v. or i.t. route. The antagonism observed was selective since the equipotent inhibition produced by the mu-opioid receptor agonist [D-Ala2, Me-Phe4, Gly(ol)5] enkephalin (DAGO) was unaffected. Overall, ICI 154,129 was considerably weaker than ICI 174,864 and both antagonists inhibited bladder activity at doses higher than those required to demonstrate delta-receptor antagonism. Further studies of the agonistic effect of ICI 174,864 showed that it was insensitive to low doses of naloxone (2 micrograms, i.c.v. or i.t.) but could be abolished by higher (10-15 micrograms) doses of naloxone. These observations suggested that the agonistic effect of ICI 174,864 was not mediated by mu-opioid receptor. beta-Endorphin (0.2-1.0 micrograms, i.c.v.) inhibited bladder contractions but following recovery from this effect, appeared to prevent the expression of delta-receptor antagonism by ICI 174,864. In addition a previously subthreshold dose of ICI 174,864 now exhibited marked agonistic

  10. Somatostatin receptor expression, tumour response, and quality of life in patients with advanced hepatocellular carcinoma treated with long-acting octreotide.

    PubMed

    Cebon, J; Findlay, M; Hargreaves, C; Stockler, M; Thompson, P; Boyer, M; Roberts, S; Poon, A; Scott, A M; Kalff, V; Garas, G; Dowling, A; Crawford, D; Ring, J; Basser, R; Strickland, A; Macdonald, G; Green, M; Nowak, A; Dickman, B; Dhillon, H; Gebski, V

    2006-10-01

    Octreotide may extend survival in hepatocellular carcinoma (HCC). Forty-one per cent of HCCs have high-affinity somatostatin receptors. We aimed to determine the feasibility, safety, and activity of long-acting octreotide in advanced HCC; to identify the best method for assessing somatostatin receptor expression; to relate receptor expression to clinical outcomes; and to evaluate toxicity. Sixty-three patients with advanced HCC received intramuscular long-acting octreotide 20 mg monthly until progression or toxicity. Median age was 67 years (range 28-81 years), male 81%, Child-Pugh A 83%, and B 17%. The aetiologies of chronic liver disease were alcohol (22%), viral hepatitis (44%), and haemochromatosis (6%). Prior treatments for HCC included surgery (8%), chemotherapy (2%), local ablation (11%), and chemoembolisation (6%). One patient had an objective partial tumour response (2%, 95% CI 0-9%). Serum alpha-fetoprotein levels decreased more than 50% in four (6%). Median survival was 8 months. Thirty four of 61 patients (56%) had receptor expression detected by scintigraphy; no clear relationship with clinical outcomes was identified. There were few grade 3 or 4 toxicities: hyperglycaemia (8%), hypoglycaemia (2%), diarrhoea (5%), and anorexia (2%). Patients reported improvements in some symptoms, but no major changes in quality of life were detected. Long-acting octreotide is safe in advanced HCC. We found little evidence of anticancer activity. A definitive randomised trial would identify whether patients benefit from this treatment in other ways. PMID:16953241

  11. Neurobiological mechanisms involved in nicotine dependence and reward: participation of the endogenous opioid system

    PubMed Central

    Berrendero, Fernando; Robledo, Patricia; Trigo, José Manuel; Martín-García, Elena; Maldonado, Rafael

    2010-01-01

    Nicotine is the primary component of tobacco that maintains the smoking habit and develops addiction. The adaptive changes of nicotinic acetylcholine receptors produced by repeated exposure to nicotine play a crucial role in the establishment of dependence. However, other neurochemical systems also participate in the addictive effects of nicotine including glutamate, cannabinoids, GABA and opioids. This review will cover the involvement of these neurotransmitters in nicotine addictive properties, with a special emphasis on the endogenous opioid system. Thus, endogenous enkephalins and beta-endorphins acting on mu-opioid receptors are involved in nicotine rewarding effects, whereas opioid peptides derived from prodynorphin participate in nicotine aversive responses. An upregulation of mu-opioid receptors has been reported after chronic nicotine treatment that could counteract the development of nicotine tolerance, whereas the downregulation induced on kappa-opioid receptors seems to facilitate nicotine tolerance. Endogenous enkephalins acting on mu-opioid receptors also play a role in the development of physical dependence to nicotine. In agreement with these actions of the endogenous opioid system, the opioid antagonist naltrexone has shown to be effective for smoking cessation in certain subpopulations of smokers. PMID:20170672

  12. Differential Stability of the Crystallographic Interfaces of Mu- and Kappa-Opioid Receptors

    PubMed Central

    Johnston, Jennifer M.; Filizola, Marta

    2014-01-01

    The recent mu-opioid receptor (MOPr) and kappa-opioid receptor (KOPr) crystal structures have inspired hypotheses of physiologically relevant dimerization contacts, specifically: a closely packed interface involving transmembrane (TM) helices TM5 and TM6, and a less compact interface, involving TM1, TM2, and helix 8 (H8). While the former was only found in MOPr crystals, similar arrangements of the latter were identified for both KOPr and MOPr. The relevance of these interfaces outside of a crystal lattice is called into question by the possibility that they might be influenced by the specific crystallization conditions. In this study, we have employed umbrella sampling molecular dynamics simulations of coarse-grained representations of the interacting MOPr or KOPr crystallographic structures, in the absence of the T4 lysozyme, and in an explicit lipid-water environment, to determine the strength of receptor dimerization at the different crystallographic interfaces. We note that the shape of the interface plays a dominant role in the strength of the interaction, and the pattern of contacting residues defines the shape of the potential of mean force. This information can be used to guide experiments aimed at exploring the role of dimerization in opioid receptor function. PMID:24651466

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

    PubMed

    Zhang, Yan; Elbegdorj, Orgil; Yuan, Yunyun; Beletskaya, Irina O; Selley, Dana E

    2013-07-01

    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 (35)S-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. PMID:23721804

  14. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques

    PubMed Central

    O’Connell, Karyn E.; Guo, Wen; Serra, Carlo; Beck, Matthew; Wachtman, Lynn; Hoggatt, Amber; Xia, Dongling; Pearson, Chris; Knight, Heather; O’Connell, Micheal; Miller, Andrew D.; Westmoreland, Susan V.; Bhasin, Shalender

    2015-01-01

    There are no approved therapies for muscle wasting in children infected with human immunodeficiency virus (HIV), which portends poor disease outcomes. To determine whether a soluble ActRIIb receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF-β/activin family members including myostatin, can prevent or restore loss of lean body mass and body weight in simian immunodeficiency virus (SIV)-infected juvenile rhesus macaques (Macaca mulatta). Fourteen pair-housed, juvenile male rhesus macaques were inoculated with SIVmac239 and, 4 wk postinoculation (WPI) treated with intramuscular injections of 10 mg ⋅ kg−1 ⋅ wk−1 ActRIIB.Fc or saline placebo. Body weight, lean body mass, SIV titers, and somatometric measurements were assessed monthly for 16 wk. Age-matched SIV-infected rhesus macaques were injected with saline. Intervention groups did not differ at baseline. Gains in lean mass were significantly greater in the ActRIIB.Fc group than in the placebo group (P < 0.001). Administration of ActRIIB.Fc was associated with greater gains in body weight (P = 0.01) and upper arm circumference than placebo. Serum CD4+ T-lymphocyte counts and SIV copy numbers did not differ between groups. Administration of ActRIIB.Fc was associated with higher muscle expression of myostatin than placebo. ActRIIB.Fc effectively blocked and reversed loss of body weight, lean mass, and fat mass in juvenile SIV-infected rhesus macaques.—O’Connell, K. E., Guo, W., Serra, C., Beck, M., Wachtman, L., Hoggatt, A., Xia, D., Pearson, C., Knight, H., O’Connell, M., Miller, A. D., Westmoreland, S. V., Bhasin, S. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques. PMID:25466897

  15. Nucleoside-derived antagonists to A3 adenosine receptors lower mouse intraocular pressure and act across species.

    PubMed

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y; Peterson-Yantorno, Kim; Stone, Richard A; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak Shin; Jacobson, Kenneth A; Civan, Mortimer M

    2010-01-01

    The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models. PMID:19878673

  16. Nucleoside-Derived Antagonists to A3 Adenosine Receptors Lower Mouse Intraocular Pressure and Act across Species

    PubMed Central

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y.; Peterson-Yantorno, Kim; Stone, Richard A.; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak Shin; Jacobson, Kenneth A.; Civan, Mortimer M.

    2009-01-01

    The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3–5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 minutes. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models. PMID:19878673

  17. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques.

    PubMed

    O'Connell, Karyn E; Guo, Wen; Serra, Carlo; Beck, Matthew; Wachtman, Lynn; Hoggatt, Amber; Xia, Dongling; Pearson, Chris; Knight, Heather; O'Connell, Micheal; Miller, Andrew D; Westmoreland, Susan V; Bhasin, Shalender

    2015-04-01

    There are no approved therapies for muscle wasting in children infected with human immunodeficiency virus (HIV), which portends poor disease outcomes. To determine whether a soluble ActRIIb receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF-β/activin family members including myostatin, can prevent or restore loss of lean body mass and body weight in simian immunodeficiency virus (SIV)-infected juvenile rhesus macaques (Macaca mulatta). Fourteen pair-housed, juvenile male rhesus macaques were inoculated with SIVmac239 and, 4 wk postinoculation (WPI) treated with intramuscular injections of 10 mg ⋅ kg(-1) ⋅ wk(-1) ActRIIB.Fc or saline placebo. Body weight, lean body mass, SIV titers, and somatometric measurements were assessed monthly for 16 wk. Age-matched SIV-infected rhesus macaques were injected with saline. Intervention groups did not differ at baseline. Gains in lean mass were significantly greater in the ActRIIB.Fc group than in the placebo group (P < 0.001). Administration of ActRIIB.Fc was associated with greater gains in body weight (P = 0.01) and upper arm circumference than placebo. Serum CD4(+) T-lymphocyte counts and SIV copy numbers did not differ between groups. Administration of ActRIIB.Fc was associated with higher muscle expression of myostatin than placebo. ActRIIB.Fc effectively blocked and reversed loss of body weight, lean mass, and fat mass in juvenile SIV-infected rhesus macaques. PMID:25466897

  18. Levodopa acts centrally to induce an antinociceptive action against colonic distension through activation of D2 dopamine receptors and the orexinergic system in the brain in conscious rats.

    PubMed

    Okumura, Toshikatsu; Nozu, Tsukasa; Kumei, Shima; Takakusaki, Kaoru; Miyagishi, Saori; Ohhira, Masumi

    2016-02-01

    Levodopa possesses antinociceptive actions against several somatic pain conditions. However, we do not know at this moment whether levodopa is also effective to visceral pain. The present study was therefore performed to clarify whether levodopa is effective to visceral pain and its mechanisms. Visceral sensation was evaluated by colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneously (80 mg/rat) or intracisternally (2.5 μg/rat) administered levodopa significantly increased the threshold of colonic distension-induced AWR in conscious rats. The dose difference to induce the antinociceptive action suggests levodopa acts centrally to exert its antinociceptive action against colonic distension. While neither sulpiride, a D2 dopamine receptor antagonist, nor SCH23390, a D1 dopamine receptor antagonist by itself changed the threshold of colonic distension-induced AWR, the intracisternally injected levodopa-induced antinociceptive action was significantly blocked by pretreatment with subcutaneously administered sulpiride but not SCH23390. Treatment with intracisternal SB334867, an orexin 1 receptor antagonist, significantly blocked the subcutaneously administered levodopa-induced antinociceptive action. These results suggest that levodopa acts centrally to induce an antinociceptive action against colonic distension through activation of D2 dopamine receptors and the orexinergic system in the brain. PMID:26883457

  19. Meta-diamide insecticides acting on distinct sites of RDL GABA receptor from those for conventional noncompetitive antagonists.

    PubMed

    Nakao, Toshifumi; Banba, Shinich; Nomura, Michikazu; Hirase, Kangetsu

    2013-04-01

    The RDL GABA receptor is an attractive target of insecticides. Here we demonstrate that meta-diamides [3-benzamido-N-(4-(perfluoropropan-2-yl)phenyl)benzamides] are a distinct class of RDL GABA receptor antagonists showing high insecticidal activity against Spodoptera litura. We also suggest that the mode of action of the meta-diamides is distinct from that of conventional noncompetitive antagonists (NCAs), such as fipronil, picrotoxin, lindane, dieldrin, and α-endosulfan. Using a membrane potential assay, we examined the effects of the meta-diamide 3-benzamido-N-(2-bromo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenyl)-2-fluorobenzamide (meta-diamide 7) and NCAs on mutant Drosophila RDL GABA receptors expressed in Drosophila Mel-2 cells. NCAs had little or no inhibitory activity against at least one of the three mutant receptors (A2'S, A2'G, and A2'N), which were reported to confer resistance to NCAs. In contrast, meta-diamide 7 inhibited all three A2' mutant receptors, at levels comparable to its activity with the wild-type receptor. Furthermore, the A2'S·T6'V mutation almost abolished the inhibitory effects of all NCAs. However, meta-diamide 7 inhibited the A2'S・T6'S mutant receptor at the same level as its activity with the wild-type receptor. In contrast, a G336M mutation in the third transmembrane domain of the RDL GABA receptor abolished the inhibitory activities of meta-diamide 7, although the G336M mutation had little effect on the inhibitory activities of conventional NCAs. Molecular modeling studies also suggested that the binding site of meta-diamides was different from those of NCAs. Meta-diamide insecticides are expected to be prominent insecticides effective against A2' mutant RDL GABA receptors with a different mode of action. PMID:23416568

  20. The phytocannabinoid, Δ9-tetrahydrocannabivarin, can act through 5-HT1A receptors to produce antipsychotic effects

    PubMed Central

    Cascio, Maria Grazia; Zamberletti, Erica; Marini, Pietro; Parolaro, Daniela; Pertwee, Roger G

    2015-01-01

    Background and Purpose This study aimed to address the questions of whether Δ9-tetrahydrocannabivarin (THCV) can (i) enhance activation of 5-HT1A receptors in vitro and (ii) induce any apparent 5-HT1A receptor-mediated antipsychotic effects in vivo. Experimental Approach In vitro studies investigated the effect of THCV on targeting by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) of 5-HT1A receptors in membranes obtained from rat brainstem or human 5-HT1A CHO cells, using [35S]-GTPγS and 8-[3H]-OH-DPAT binding assays. In vivo studies investigated whether THCV induces signs of 5-HT1A receptor-mediated antipsychotic effects in rats. Key Results THCV (i) potently, albeit partially, displaced 8-[3H]-OH-DPAT from specific binding sites in rat brainstem membranes; (ii) at 100 nM, significantly enhanced 8-OH-DPAT-induced activation of receptors in these membranes; (iii) produced concentration-related increases in 8-[3H]-OH-DPAT binding to specific sites in membranes of human 5-HT1A receptor-transfected CHO cells; and (iv) at 100 nM, significantly enhanced 8-OH-DPAT-induced activation of these human 5-HT1A receptors. In phencyclidine-treated rats, THCV, like clozapine (i) reduced stereotyped behaviour; (ii) decreased time spent immobile in the forced swim test; and (iii) normalized hyperlocomotor activity, social behaviour and cognitive performance. Some of these effects were counteracted by the 5-HT1A receptor antagonist, WAY100635, or could be reproduced by the CB1 antagonist, AM251. Conclusions and Implications Our findings suggest that THCV can enhance 5-HT1A receptor activation, and that some of its apparent antipsychotic effects may depend on this enhancement. We conclude that THCV has therapeutic potential for ameliorating some of the negative, cognitive and positive symptoms of schizophrenia. PMID:25363799

  1. Gender Interacts with Opioid Receptor Polymorphism A118G and Serotonin Receptor Polymorphism -1438 A/G on Speed-Dating Success.

    PubMed

    Wu, Karen; Chen, Chuansheng; Moyzis, Robert K; Greenberger, Ellen; Yu, Zhaoxia

    2016-09-01

    We examined an understudied but potentially important source of romantic attraction-genetics-using a speed-dating paradigm. The mu opioid receptor (OPRM1) polymorphism A118G (rs1799971) and the serotonin receptor (HTR2A) polymorphism -1438 A/G (rs6311) were studied because they have been implicated in social affiliation. Guided by the social role theory of mate selection and prior genetic evidence, we examined these polymorphisms' gender-specific associations with speed-dating success (i.e., date offers, mate desirability). A total of 262 single Asian Americans went on speed-dates with members of the opposite gender and completed interaction questionnaires about their partners. Consistent with our prediction, significant gender-by-genotype interactions were found for speed-dating success. Specifically, the minor variant of A118G (G-allele), which has been linked to submissiveness/social sensitivity, predicted greater speed-dating success for women, whereas the minor variant of -1438 A/G (G-allele), which has been linked to leadership/social dominance, predicted greater speed-dating success for men. For both polymorphisms, reverse "dampening" effects of minor variants were found for opposite-gender counterparts. These results support previous research on the importance of the opioid and serotonergic systems in social affiliation, indicating that their influence extends to dating success, with opposite, yet gender-norm consistent, effects for men and women. PMID:27193909

  2. Monoamine receptor agonists, acting preferentially at presynaptic autoreceptors and heteroreceptors, downregulate the cell fate adaptor FADD in rat brain cortex.

    PubMed

    García-Fuster, M Julia; García-Sevilla, Jesús A

    2015-02-01

    FADD is a crucial adaptor of death receptors that can engage apoptosis or survival actions (e.g. neuroplasticity) through its phosphorylated form (p-FADD). Although FADD was shown to participate in receptor mechanisms related to drugs of abuse, little is known on its role in the signaling of classic neurotransmitters (dopamine, noradrenaline, and serotonin) in brain. This study assessed the modulation of FADD (and p-FADD/FADD ratio, as an index of neuroplasticity) and FLIP-L (a neuroprotective FADD interacting partner), as well as the role of MEK-ERK signaling, after activation of monoamine auto/heteroreceptors by selective agonists in rat cortex. Acute depletion of monoamines with reserpine, but not with AMPT or PCPA, reduced FADD (28%) and increased p-FADD/FADD ratio (1.34-fold). Activation of presynaptic α2A-adrenoceptors (UK-14304 and clonidine), 5-HT1A receptors (8-OH-DPAT), and D2 dopamine receptor (bromocriptine) dose-dependently decreased FADD (up to 54%) and increased p-FADD (up to 29%) and p-FADD/FADD ratios (up to 2.93-fold), through specific receptor mechanisms. Activation of rat 5-HT1B autoreceptor in axon terminals by CP-94253 did not modulate FADD forms. Activation of postsynaptic D1 dopamine receptor by SKF-81297 also reduced FADD (25%) and increased p-FADD (32%). Disruption of MEK-ERK activation with SL327 did not modify clonidine (α2A-adrenoceptor)-induced FADD inhibition, indicating that agonist effect was not dependent on ERK signaling. The various monoamine receptor agonists and antagonists did not alter FLIP-L content, or the activation of executioner caspase-3 and PARP-1 cleavage, indicating that the agonists attenuated apoptotic signals and promoted neuroplasticity through FADD regulation. These novel results indicate that inhibition of pro-apoptotic FADD adaptor could function as a common signaling step in the initial activation of monoamine receptors in the brain. PMID:25286119

  3. Heat shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells.

    PubMed

    Wampler, Jennifer L; Kim, Kwang-Pyo; Jaradat, Ziad; Bhunia, Arun K

    2004-02-01

    The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion. PMID:14742538

  4. Frienemies of infection: A chronic case of host nuclear receptors acting as cohorts or combatants of infection.

    PubMed

    Mahajan, Sahil; Saini, Ankita; Kalra, Rashi; Gupta, Pawan

    2016-08-01

    Macrophages and dendritic cells provide critical effector functions to efficiently resist and promptly eliminate infection. Pattern recognition receptors signaling operative in these cell types is imperative for their innate properties. However, it is now emerging that besides these conventional signaling pathways, nuclear receptors coupled gene regulation and transrepression pathways assemble immune regulatory networks. A couple of these networks associated with members of nuclear receptor superfamily decide heterogeneity in macrophages and dendritic cells population and thereby play decisive role in determining protective immunity against bacteria, viruses, fungi, protozoa and helminths. Pathogens also direct shift in the expression of nuclear receptors and their target genes and this is proclaimed to be a sui generis mechanism whereby microbes disconnect the genomic component from the peripheral immune response. Many endogenous and synthetic nuclear receptor ligands have been tested in various in vitro and in vivo infection models to study their effect on pathogen burden. Here, we discuss current advances in our understanding of the composite interactions between nuclear receptor and pathogens and their implications on the causatum infectious diseases. PMID:25358058

  5. Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

    PubMed Central

    Dorfman, Mauricio D.; Kerr, Bredford; Garcia-Rudaz, Cecilia; Paredes, Alfonso H.; Dissen, Gregory A.

    2011-01-01

    Tropomyosin-related kinase (TRK) receptor B (TRKB) mediates the supportive actions of neurotrophin 4/5 and brain-derived neurotrophic factor on early ovarian follicle development. Absence of TRKB receptors reduces granulosa cell (GC) proliferation and delays follicle growth. In the present study, we offer mechanistic insights into this phenomenon. DNA array and quantitative PCR analysis of ovaries from TrkB-null mice revealed that by the end of the first week of postnatal life, Jagged1, Hes1, and Hey2 mRNA abundance is reduced in the absence of TRKB receptors. Although Jagged1 encodes a NOTCH receptor ligand, Hes1 and Hey2 are downstream targets of the JAGGED1-NOTCH2 signaling system. Jagged1 is predominantly expressed in oocytes, and the abundance of JAGGED1 is decreased in TrkB−/− oocytes. Lack of TRKB receptors also resulted in reduced expression of c-Myc, a NOTCH target gene that promotes entry into the cell cycle, but did not alter the expression of genes encoding core regulators of cell-cycle progression. Selective restoration of JAGGED1 synthesis in oocytes of TrkB−/− ovaries via lentiviral-mediated transfer of the Jagged1 gene under the control of the growth differentiation factor 9 (Gdf9) promoter rescued c-Myc expression, GC proliferation, and follicle growth. These results suggest that neurotrophins acting via TRKB receptors facilitate early follicle growth by supporting a JAGGED1-NOTCH2 oocyte-to-GC communication pathway, which promotes GC proliferation via a c-MYC-dependent mechanism. PMID:22028443

  6. Nascent structure-activity relationship study of a diastereomeric series of kappa opioid receptor antagonists derived from CJ-15,208.

    PubMed

    Dolle, Roland E; Michaut, Mathieu; Martinez-Teipel, Blanca; Seida, Pamela R; Ajello, Christopher W; Muller, Alison L; DeHaven, Robert N; Carroll, Patrick J

    2009-07-01

    Cyclic tetrapeptide c[Phe-pro-Phe-trp] 2, a diastereomer of CJ-15,208 (1), was identified as a potent dual kappa/mu opioid receptor antagonist devoid of delta opioid receptor affinity against cloned human receptors: K(i) (2)=3.8nM (kappa), 30nM (mu); IC(50) ([(35)S]GTPgammaS binding)=140nM (kappa), 21nM (mu). The d-tryptophan residue rendered 2 ca. eightfold and fourfold more potent at kappa and mu, respectively, than the corresponding l-configured tryptophan in the natural product 1. Phe analogs 3-10, designed to probe the effect of substituents on receptor affinity and selectivity, possessed K(i) values ranging from 14 to 220nM against the kappa opioid receptor with mu/kappa ratios of 0.45-3.0. An alanine scan of 2 yielded c[Ala-pro-Phe-trp] 12, an analog equipotent to 2. Agents 2 and 12 were pure antagonists in vitro devoid of agonist activity. Ac-pro-Phe-trp-Phe-NH(2)16 and Ac-Phe-trp-Phe-pro-NH(2)17 two of the eight possible acyclic peptides derived from 1 and 2, were selective, modestly potent mu ligands: K(i) (16)=340nM (mu); K(i) (17)=360nM (mu). PMID:19464172

  7. D1-type dopamine receptors inhibit growth cone motility in cultured retina neurons: evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system.

    PubMed Central

    Lankford, K L; DeMello, F G; Klein, W L

    1988-01-01

    Precedent exists for the early development and subsequent down-regulation of neurotransmitter receptor systems in the vertebrate central nervous system, but the function of such embryonic receptors has not been established. Here we show that stimulation of early-developing dopamine receptors in avian retina cells greatly inhibits the motility of neuronal growth cones. Neurons from embryonic chicken retinas were cultured in low-density monolayers, and their growth cones were observed with phase-contrast or video-enhanced-contrast-differential-interference-contrast (VEC-DIC) microscopy. Approximately 25% of the neurons responded to micromolar dopamine with a rapid reduction in filopodial activity followed by a flattening of growth cones and retraction of neurites. The response occurred at all ages examined (embryonic day-8 retinal neurons cultured on polylysine-coated coverslips for 1-7 days), although neurite retraction was greatest in younger cultures. Effects of dopamine on growth cone function could be reversed by haloperidol or (+)-SCH 23390, whereas forskolin elicited a response similar to dopamine; these data show the response was receptor-mediated, acting through a D1-type system, and are consistent with the use of cAMP as a second messenger. The experiments provide strong support for the hypothesis that neurotransmitters, besides mediating transynaptic signaling in the adult, may have a role in neuronal differentiation as growth regulators. Images PMID:3380807

  8. D1-type dopamine receptors inhibit growth cone motility in cultured retina neurons: evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system.

    PubMed Central

    Lankford, K L; DeMello, F G; Klein, W L

    1988-01-01

    Precedent exists for the early development and subsequent down-regulation of neurotransmitter receptor systems in the vertebrate central nervous system, but the function of such embryonic receptors has not been established. Here we show that stimulation of early-developing dopamine receptors in avian retina cells greatly inhibits the motility of neuronal growth cones. Neurons from embryonic chicken retinas were cultured in low-density monolayers, and their growth cones were observed with phase-contrast or video-enhanced-contrast-differential-interference-contrast (VEC-DIC) microscopy. Approximately 25% of the neurons responded to micromolar dopamine with a rapid reduction in filopodial activity followed by a flattening of growth cones and retraction of neurites. The response occurred at all ages examined (embryonic day-8 retinal neurons cultured on polylysine-coated coverslips for 1-7 days), although neurite retraction was greatest in younger cultures. Effects of dopamine on growth cone function could be reversed by haloperidol or (+)-SCH 23390, whereas forskolin elicited a response similar to dopamine; these data show the response was receptor-mediated, acting through a D1-type system, and are consistent with the use of cAMP as a second messenger. The experiments provide strong support for the hypothesis that neurotransmitters, besides mediating transynaptic signaling in the adult, may have a role in neuronal differentiation as growth regulators. Images PMID:3357895

  9. The immune receptor Tim-3 acts as a trafficker in a Tim-3/galectin-9 autocrine loop in human myeloid leukemia cells.

    PubMed

    Gonçalves Silva, Isabel; Rüegg, Laura; Gibbs, Bernhard F; Bardelli, Marco; Fruehwirth, Alexander; Varani, Luca; Berger, Steffen M; Fasler-Kan, Elizaveta; Sumbayev, Vadim V

    2016-07-01

    The immune receptor Tim-3 is often highly expressed in human acute myeloid leukemia (AML) cells where it acts as a growth factor and inflammatory receptor. Recently, it has been demonstrated that Tim-3 forms an autocrine loop with its natural ligand galectin-9 in human AML cells. However, the pathophysiological functions of Tim-3 in human AML cells remain unclear. Here, we report for the first time that Tim-3 is required for galectin-9 secretion in human AML cells. However, this effect is cell-type specific and was found so far to be applicable only to myeloid (and not, for example, lymphoid) leukemia cells. We concluded that AML cells might use Tim-3 as a trafficker for the secretion of galectin-9 which can then be possibly used to impair the anticancer activities of cytotoxic T cells and natural killer (NK) cells. PMID:27622049

  10. The immune receptor Tim-3 acts as a trafficker in a Tim-3/galectin-9 autocrine loop in human myeloid leukemia cells

    PubMed Central

    Gonçalves Silva, Isabel; Rüegg, Laura; Gibbs, Bernhard F.; Bardelli, Marco; Fruehwirth, Alexander; Varani, Luca; Berger, Steffen M.; Fasler-Kan, Elizaveta; Sumbayev, Vadim V.

    2016-01-01

    ABSTRACT The immune receptor Tim-3 is often highly expressed in human acute myeloid leukemia (AML) cells where it acts as a growth factor and inflammatory receptor. Recently, it has been demonstrated that Tim-3 forms an autocrine loop with its natural ligand galectin-9 in human AML cells. However, the pathophysiological functions of Tim-3 in human AML cells remain unclear. Here, we report for the first time that Tim-3 is required for galectin-9 secretion in human AML cells. However, this effect is cell-type specific and was found so far to be applicable only to myeloid (and not, for example, lymphoid) leukemia cells. We concluded that AML cells might use Tim-3 as a trafficker for the secretion of galectin-9 which can then be possibly used to impair the anticancer activities of cytotoxic T cells and natural killer (NK) cells. PMID:27622049

  11. Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors.

    PubMed

    Falcon, Edgardo; Browne, Caroline A; Leon, Rosa M; Fleites, Vanessa C; Sweeney, Rachel; Kirby, Lynn G; Lucki, Irwin

    2016-08-01

    Previous studies have identified potential antidepressant effects of buprenorphine (BPN), a drug with high affinity for mu opioid receptor (MORs) and kappa opioid receptors (KORs) and some affinity at delta opioid receptor (DOR) and opioid receptor-like 1 (ORL-1) receptors. Therefore, these studies examined which opioid receptors were involved in BPN's effects on animal behavior tests sensitive to antidepressant drugs. The acute effects of BPN were tested in the forced swim test (FST) using mice with genetic deletion of individual opioid receptors or after pharmacological blockade of receptors. For evaluating the effects of BPN on chronic stress, separate groups of mice were exposed to unpredictable chronic mild stress (UCMS) for 3 weeks and treated with BPN for at least 7 days before behavioral assessment and subsequent measurement of Oprk1, Oprm1, and Pdyn mRNA expression in multiple brain regions. BPN did not reduce immobility in mice with KOR deletion or after pretreatment with norbinaltorphimine, even though desipramine remained effective. In contrast, BPN reduced immobility in MOR and DOR knockout mice and in mice pretreated with the ORL-1 antagonist JTC-801. UCMS reduced sucrose preference, decreased time in the light side of the light/dark box, increased immobility in the FST and induced region-specific alterations in Oprk1, Oprm1, and PDYN mRNA expression in the frontal cortex and striatum. All of these changes were normalized following BPN treatment. The KOR was identified as a key player mediating the effects of BPN in tests sensitive to antidepressant drugs in mice. These studies support further development of BPN as a novel antidepressant. PMID:26979295

  12. The selective orexin receptor 1 antagonist ACT-335827 in a rat model of diet-induced obesity associated with metabolic syndrome.

    PubMed

    Steiner, Michel A; Sciarretta, Carla; Pasquali, Anne; Jenck, Francois

    2013-01-01

    The orexin system regulates feeding, nutrient metabolism and energy homeostasis. Acute pharmacological blockade of orexin receptor 1 (OXR-1) in rodents induces satiety and reduces normal and palatable food intake. Genetic OXR-1 deletion in mice improves hyperglycemia under high-fat (HF) diet conditions. Here we investigated the effects of chronic treatment with the novel selective OXR-1 antagonist ACT-335827 in a rat model of diet-induced obesity (DIO) associated with metabolic syndrome (MetS). Rats were fed either standard chow (SC) or a cafeteria (CAF) diet comprised of intermittent human snacks and a constant free choice between a HF/sweet (HF/S) diet and SC for 13 weeks. Thereafter the SC group was treated with vehicle (for 4 weeks) and the CAF group was divided into a vehicle and an ACT-335827 treatment group. Energy and water intake, food preference, and indicators of MetS (abdominal obesity, glucose homeostasis, plasma lipids, and blood pressure) were monitored. Hippocampus-dependent memory, which can be impaired by DIO, was assessed. CAF diet fed rats treated with ACT-335827 consumed less of the HF/S diet and more of the SC, but did not change their snack or total kcal intake compared to vehicle-treated rats. ACT-335827 increased water intake and the high-density lipoprotein associated cholesterol proportion of total circulating cholesterol. ACT-335827 slightly increased body weight gain (4% vs. controls) and feed efficiency in the absence of hyperphagia. These effects were not associated with significant changes in the elevated fasting glucose and triglyceride (TG) plasma levels, glucose intolerance, elevated blood pressure, and adiposity due to CAF diet consumption. Neither CAF diet consumption alone nor ACT-335827 affected memory. In conclusion, the main metabolic characteristics associated with DIO and MetS in rats remained unaffected by chronic ACT-335827 treatment, suggesting that pharmacological OXR-1 blockade has minimal impact in this model. PMID

  13. The selective orexin receptor 1 antagonist ACT-335827 in a rat model of diet-induced obesity associated with metabolic syndrome

    PubMed Central

    Steiner, Michel A.; Sciarretta, Carla; Pasquali, Anne; Jenck, Francois

    2013-01-01

    The orexin system regulates feeding, nutrient metabolism and energy homeostasis. Acute pharmacological blockade of orexin receptor 1 (OXR-1) in rodents induces satiety and reduces normal and palatable food intake. Genetic OXR-1 deletion in mice improves hyperglycemia under high-fat (HF) diet conditions. Here we investigated the effects of chronic treatment with the novel selective OXR-1 antagonist ACT-335827 in a rat model of diet-induced obesity (DIO) associated with metabolic syndrome (MetS). Rats were fed either standard chow (SC) or a cafeteria (CAF) diet comprised of intermittent human snacks and a constant free choice between a HF/sweet (HF/S) diet and SC for 13 weeks. Thereafter the SC group was treated with vehicle (for 4 weeks) and the CAF group was divided into a vehicle and an ACT-335827 treatment group. Energy and water intake, food preference, and indicators of MetS (abdominal obesity, glucose homeostasis, plasma lipids, and blood pressure) were monitored. Hippocampus-dependent memory, which can be impaired by DIO, was assessed. CAF diet fed rats treated with ACT-335827 consumed less of the HF/S diet and more of the SC, but did not change their snack or total kcal intake compared to vehicle-treated rats. ACT-335827 increased water intake and the high-density lipoprotein associated cholesterol proportion of total circulating cholesterol. ACT-335827 slightly increased body weight gain (4% vs. controls) and feed efficiency in the absence of hyperphagia. These effects were not associated with significant changes in the elevated fasting glucose and triglyceride (TG) plasma levels, glucose intolerance, elevated blood pressure, and adiposity due to CAF diet consumption. Neither CAF diet consumption alone nor ACT-335827 affected memory. In conclusion, the main metabolic characteristics associated with DIO and MetS in rats remained unaffected by chronic ACT-335827 treatment, suggesting that pharmacological OXR-1 blockade has minimal impact in this model. PMID

  14. Identification of small molecule inhibitors of amyloid β-induced neuronal apoptosis acting through the imidazoline I(2) receptor.

    PubMed

    Montolio, Marisol; Gregori-Puigjané, Elisabet; Pineda, David; Mestres, Jordi; Navarro, Pilar

    2012-11-26

    Aberrant activation of signaling pathways plays a pivotal role in central nervous system disorders, such as Alzheimer's disease (AD). Using a combination of virtual screening and experimental testing, novel small molecule inhibitors of tPA-mediated extracellular signal-regulated kinase (Erk)1/2 activation were identified that provide higher levels of neuroprotection from Aβ-induced apoptosis than Memantine, the most recently FDA-approved drug for AD treatment. Subsequent target deconvolution efforts revealed that they all share low micromolar affinity for the imidazoline I(2) receptor, while being devoid of any significant affinity to a list of AD-relevant targets, including the N-methyl-d-aspartate receptor (NMDAR), acetylcholinesterase (AChE), and monoamine oxidase B (MAO-B). Targeting the imidazoline I(2) receptor emerges as a new mechanism of action to inhibit tPA-induced signaling in neurons for the treatment of AD and other neurodegenerative diseases. PMID:23098038

  15. Adenosine trisphosphate appears to act via different receptors in terminals versus somata of the hypothalamic neurohypophysial system.

    PubMed

    Knott, T K; Hussy, N; Cuadra, A E; Lee, R H; Ortiz-Miranda, S; Custer, E E; Lemos, J R

    2012-04-01

    ATP-induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC(50) of 50 μm in somata versus 9.6 μm in terminals). The ATP-induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC(50) of 3.6 μm in somata versus 11.6 μm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP-γ-S and benzoyl-benzoyl-ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC(50) , inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones. PMID:22340013

  16. The Rapidly Acting Antidepressant Ketamine and the mGlu2/3 Receptor Antagonist LY341495 Rapidly Engage Dopaminergic Mood Circuits.

    PubMed

    Witkin, J M; Monn, J A; Schoepp, D D; Li, X; Overshiner, C; Mitchell, S N; Carter, G; Johnson, B; Rasmussen, K; Rorick-Kehn, L M

    2016-07-01

    Ketamine is a rapidly acting antidepressant in patients with treatment-resistant depression (TRD). Although the mechanisms underlying these effects are not fully established, inquiry to date has focused on the triggering of synaptogenesis transduction pathways via glutamatergic mechanisms. Preclinical data suggest that blockade of metabotropic glutamate (mGlu2/3) receptors shares many overlapping features and mechanisms with ketamine and may also provide rapid efficacy for TRD patients. Central dopamine circuitry is recognized as an end target for mood regulation and hedonic valuation and yet has been largely neglected in mechanistic studies of antidepressant-relevant effects of ketamine. Herein, we evaluated the changes in dopaminergic neurotransmission after acute administration of ketamine and the mGlu2/3 receptor antagonist LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid ] in preclinical models using electrophysiologic, neurochemical, and behavioral endpoints. When given acutely, both ketamine and LY341495, but not the selective serotonin reuptake inhibitor (SSRI) citalopram, increased the number of spontaneously active dopamine neurons in the ventral tegmental area (VTA), increased extracellular levels of dopamine in the nucleus accumbens and prefrontal cortex, and enhanced the locomotor stimulatory effects of the dopamine D2/3 receptor agonist quinpirole. Further, both ketamine and LY341495 reduced immobility time in the tail-suspension assay in CD1 mice, which are relatively resistant to SSRI antidepressants. Both the VTA neuronal activation and the antidepressant phenotype induced by ketamine and LY341495 were attenuated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo- (9CI)-benzo[f]quinoxaline-7-sulfonamide, indicating AMPA-dependent effects. These findings provide another overlapping mechanism of action of ketamine and mGlu2/3 receptor

  17. Analgesia produced by exposure to 2450-MHz radiofrequency radiation (RFR) is mediated by brain mu- and kappa-opioid receptors

    SciTech Connect

    Salomon, G.; Park, E.J.; Quock, R.M. )

    1992-02-26

    This study was conducted to identify the opioid receptor subtype(s) responsible for RFR-induced analgesia. Male Swiss Webster mice, 20-25 g, were exposed to 20 mW/cm{sup 2} RFR in a 2,450-MHz waveguide system for 10 min, then tested 15 min later in the abdominal constriction paradigm which detects {mu}- and {kappa}-opioid activity. Immediately following RFR exposure, different groups of mice were pretreated intracerebroventricularly with different opioid receptor blockers with selectivity for {mu}- or {kappa}-opioid receptors. Results show that RFR-induced analgesia was attenuated by higher but not lower doses of the non-selective antagonist naloxone, but the selective {mu}-opioid antagonist {beta}-funaltrexamine and by the selective {kappa}-opioid antagonist norbinaltorphimine. RFR-induced analgesia was also reduced by subcutaneous pretreatment with 5.0 mg/kg of the {mu}-/{kappa}-opioid antagonist({minus})-5,9-diethyl-{alpha}-5,9-dialkyl-2{prime}-hydroxy-6,7-benzomorphan(MR-2266). These findings suggest that RFR-induced analgesia may be mediated by both {mu}- and {kappa}-opioid mechanisms.

  18. Sex-related differences in mechanical nociception and antinociception produced by mu- and kappa-opioid receptor agonists in rats.

    PubMed

    Barrett, Andrew C; Smith, Eric S; Picker, Mitchell J

    2002-10-01

    Previous studies indicate that in antinociceptive procedures employing thermal, chemical and electrical stimuli, opioids are generally more potent in male than female rodents. The purpose of the present study was to examine nociception and opioid antinociception in male and female rats using a mechanical nociceptive stimulus. Results indicated that males had a higher threshold for nociception, and in tests in which a constant pressure was applied to the hindpaw, the paw withdrawal latencies were consistently longer in males. Opioids with activity at the mu receptor, including levorphanol, morphine, dezocine, buprenorphine, butorphanol and nalbuphine, were generally more potent and/or effective in males. In contrast, sex differences were not consistently observed with the kappa-opioid receptor agonists spiradoline, (5,7,8b)-N-methyl-N[2-1(1-pyrrolidinyl),1-oxaspiro[4,5]dec-8-yl benzeneacetamide (U69593), trans-(+/-)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50488), enadoline, ethylketocyclazocine, and nalorphine. These findings suggest that males and females differ in their responsiveness to mechanical nociception and that sex differences in sensitivity to kappa-, but not mu-, opioid receptor agonists are specific to certain nociceptive stimulus modalities. PMID:12354566

  19. Functional evidence for multiple receptor activation by kappa-ligands in the inhibition of spinal nociceptive reflexes in the rat.

    PubMed Central

    Herrero, J. F.; Headley, P. M.

    1993-01-01

    1. The evidence for kappa-receptor heterogeneity is equivocal. We have now investigated this question by comparing the effects of five putatively selective kappa-agonists. The parameters examined were: the relative potencies in depressing hindlimb flexor muscle reflexes to noxious pinch stimuli in both spinalized and sham-spinalized rats; the reversibility of these effects by naloxone; and the effects on blood pressure. 2. Two types of drug effect was discriminated. One drug group, represented by U-50,488, U-69,593 and PD-117,302, had a potency ratio between sham and spinalized rats approximately 10 fold lower than the other group, which comprised GR103545 and CI-977. 3. Under sham-spinalized conditions, CI-977 and GR103545 at high doses caused only sub-maximal reductions of spinal reflexes. U-50,488 was still active when superimposed on these high doses of GR103545. 4. Naloxone reversed all effects, but different doses were required between compounds, with GR103545 taking some 20 times higher doses of naloxone to cause reversal than did U-50,488. 5. The effects on mean arterial pressure were opposite between groups. 6. The results imply that more than one type of naloxone-sensitive non-mu opioid receptor must be involved in mediating these complex actions of ligands that have been claimed to be selective for kappa-receptors. PMID:8220893

  20. Host cell heparan sulfate proteoglycans mediate attachment and entry of Listeria monocytogenes, and the listerial surface protein ActA is involved in heparan sulfate receptor recognition.

    PubMed

    Alvarez-Domínguez, C; Vázquez-Boland, J A; Carrasco-Marín, E; López-Mato, P; Leyva-Cobián, F

    1997-01-01

    The mechanisms by which the intracellular pathogen Listeria monocytogenes interacts with the host cell surface remain largely unknown. In this study, we investigated the role of heparan sulfate proteoglycans (HSPG) in listerial infection. Pretreatment of bacteria with heparin or heparan sulfate (HS), but not with other glycosaminoglycans, inhibited attachment and subsequent uptake by IC-21 murine macrophages and CHO epithelial-like cells. Specific removal of HS from target cells with heparinase III significantly impaired listerial adhesion and invasion. Mutant CHO cells deficient in HS synthesis bound and internalized significantly fewer bacteria than wild-type cells did. Pretreatment of target cells with the HS-binding proteins fibronectin and platelet factor 4, or with heparinase III, impaired listerial infectivity only in those cells expressing HS. Moreover, a synthetic peptide corresponding to the HS-binding ligand in Plasmodium falciparum circumsporozoite protein (pepPf1) inhibited listerial attachment to IC-21 and CHO cells. A motif very similar to the HS-binding site of pepPf1 was found in the N-terminal region of ActA, the L. monocytogenes surface protein responsible for actin-based bacterial motility and cell-to-cell spread. In the same region of ActA, several clusters of positively charged amino acids which could function as HS-binding domains were identified. An ActA-deficient mutant was significantly impaired in attachment and entry due to altered HS recognition functions. This work shows that specific interaction with an HSPG receptor present on the surface of both professional and nonprofessional phagocytes is involved in L. monocytogenes cytoadhesion and invasion and strongly suggests that the bacterial surface protein ActA may be a ligand mediating HSPG receptor recognition. PMID:8975895

  1. The terpenoids Myrtenol and Verbenol act on δ subunit-containing GABAA receptors and enhance tonic inhibition in dentate gyrus granule cells.

    PubMed

    van Brederode, Johannes; Atak, Sinem; Kessler, Artur; Pischetsrieder, Monika; Villmann, Carmen; Alzheimer, Christian

    2016-08-15

    Sideritis plants and their extracts have been used in traditional medicine as sedatives, anxiolytics and anticonvulsant agents. Pinenes are the most prevalent of the volatile aroma components in Siderites extracts and the pinene metabolites myrtenol and verbenol have been identified as the most potent positive allosteric modulators of synaptic GABAA receptors composed of α1β2 and α1β2γ2 subunits. In view of their therapeutic spectrum, we wondered whether these two terpenoids would also augment tonic GABA currents mediated by extrasynaptic GABAA receptors containing the δ subunit. When we expressed α4β2δ receptors in HEK293 cells, we found that co-application of myrtenol or verbenol enhanced whole-cell current responses to GABA by up to 100%. Consistent with their effects on heterologous α1β2γ2 receptors, we found that myrtenol and verbenol, when co-applied with GABA via local perfusion, increased the amplitude and area of miniature inhibitory postsynaptic potentials (mIPSCs) recorded in whole-cell voltage-clamp recordings from granule cells in the dentate gyrus of mouse hippocampal brain slices. In addition, co-application of terpenoids with GABA was also able to enhance tonic GABA current, measured from the change in baseline current and current noise, compared to GABA perfusion alone. Our results suggest that myrtenol and verbenol act as positive allosteric modulators at synaptic and extrasynaptic GABAA receptors, thereby augmenting phasic and tonic GABAergic inhibition. Thus, our study reveals an important pharmacological and therapeutic target of bicyclic monoterpenoids. PMID:27312536

  2. ROLES OF OPIOID RECEPTOR SUBTYPES IN MEDIATING ALCOHOL SEEKING INDUCED BY DISCRETE CUES AND CONTEXT

    PubMed Central

    Marinelli, Peter W.; Funk, Douglas; Harding, Stephen; Li, Zhaoxia; Juzytsch, Walter; Lê, A.D.

    2009-01-01

    The aim of this study was to assess the effects of selective blockade of the delta (DOP) or mu opioid (MOP) receptors on alcohol seeking induced by discrete cues and context. In Experiment 1, rats were trained to self-administer alcohol in an environment with distinct sensory properties. After extinction in a different context with separate sensory properties, rats were tested for context-induced renewal in the original context following treatment with the DOP receptor antagonist naltrindole (0 – 15-mg/kg, IP) or the MOP receptor antagonist CTOP (0 – 3-µg/kg ICV). In a separate set of experiments, reinstatement was tested with the presentation of a discrete light+tone cue previously associated with alcohol delivery, following extinction without the cue. In Experiment 2, the effects of naltrindole (0 – 5-mg/kg, IP) or CTOP (0 – 3-µg/kg µg ICV) were assessed. For context-induced renewal, 7.5-mg/kg naltrindole reduced responding without affecting locomotor activity. Both doses of CTOP attenuated responding in the first 15 min of the renewal test session; however, total responses did not differ at the end of the session. For discrete cue-induced reinstatement, 1 and 5-mg/kg naltrindole attenuated responding, but CTOP had no effect. We conclude that while DOP receptors mediate alcohol seeking induced by discrete cues and context, MOP receptors may play a modest role only in context-induced renewal. These findings point to a differential involvement of opioid receptor subtypes in the effects of different kinds of conditioned stimuli on alcohol seeking, and support a more prominent role for DOP receptors. PMID:19686472

  3. Role of preoptic opioid receptors in the body temperature reduction during hypoxia.

    PubMed

    Scarpellini, Carolina da Silveira; Gargaglioni, Luciane H; Branco, Luis G S; Bícego, Kênia C

    2009-08-25

    Evidence indicates that endogenous opioids play a role in body temperature (Tb) regulation in mammals but no data exist about the involvement of the specific opioid receptors, mu, kappa and delta, in the reduction of Tb induced by hypoxia. Thus, we investigated the participation of these opioid receptors in the anteroventral preoptic region (AVPO) in hypoxic decrease of Tb. To this end, Tb of unanesthetized Wistar rats was monitored by temperature data loggers before and after intra-AVPO microinjection of the selective kappa-opioid receptor antagonist nor-binaltorphimine dihydrochloride (nor-BNI; 0.1 and 1.0 microg/100 nL/animal), the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 cyclic (CTAP; 0.1 and 1.0 microg/100 nL/animal), and the selective delta-opioid receptor antagonist Naltrindole (0.06 and 0.6 microg/100 nL/animal) or saline (vehicle, 100 nL/animal), during normoxia and hypoxia (7% inspired O2). Under normoxia, no effect of opioid antagonists on Tb was observed. Hypoxia induced Tb to reduce in vehicle group, a response that was inhibited by the microinjection intra-AVPO of nor-BNI. In contrast, CTAP and Naltrindole did not change Tb during hypoxia but caused a longer latency for the return of Tb to the normoxic values just after low O2 exposure. Our results indicate the kappa-opioid receptor in the AVPO is important for the reduction of Tb during hypoxia while the mu and delta receptors are involved in the increase of Tb during normoxia post-hypoxia. PMID:19545549

  4. Increased adiposity on normal diet, but decreased susceptibility to diet-induced obesity in μ-opioid receptor-deficient mice

    PubMed Central

    Zuberi, Aamir R.; Townsend, Leigh; Patterson, Laurel; Zheng, Huiyuan; Berthoud, Hans-Rudi

    2008-01-01

    The mu-opioid receptor encoded by the Oprm1 gene plays a crucial role in the mediation of food reward and drug-induced positive reinforcement, but its genetic deletion has been shown to provide food intake-independent, partial protection from diet-induced obesity. We hypothesized that mu-opioid receptor-deficient mice would show an even greater, intake-dependent, resistance to high fat diet-induced obesity if the diet comprises a sweet component. We generated an F2 population by crossing the heterozygous offspring of homozygous female Oprm1-/- mice (on a mixed C57BL/6 and BALB/c genetic background) with male inbred C57BL/6 mice. Groups of genotyped wildtype (WT) and homozygous mutant (KO) males and females were fed either control chow or a high caloric palatable diet consisting of sweet, liquid chocolate-flavored Ensure together with a solid high fat diet. Food intake, body weight, and body composition was measured over a period of 16 weeks. Unexpectedly, male, and to a lesser extent female, KO mice fed chow for the entire period showed progressively increased body weight and adiposity while eating significantly more chow. In contrast, when exposed to the sweet plus high-fat diet, male, and to a lesser extent female, KO mice gained significantly less body weight and fat mass compared to WT mice when using chow fed counterparts for reference values. Male KO mice consumed 33% less of the sweet liquid diet but increased intake of high-fat pellets, so that total calorie intake was not different from WT animals. These results demonstrate a dissociation of the role of μ-opioid receptors in the control of adiposity for different diets and sex. On a bland diet, normal receptor function appears to confer a slightly catabolic predisposition, but on a highly palatable diet, it confers an anabolic metabolic profile, favoring fat accretion. Because of the complexity of μ-opioid gene regulation and tissue distribution, more selective and targeted approaches will be necessary

  5. A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain

    PubMed Central

    Andó, RD; Méhész, B; Gyires, K; Illes, P; Sperlágh, B

    2010-01-01

    Background and purpose: This study was undertaken to compare the analgesic activity of antagonists acting at P2X1, P2X7, and P2Y12 receptors and agonists acting at P2Y1, P2Y2, P2Y4, and P2Y6 receptors in neuropathic, acute, and inflammatory pain. Experimental approach: The effect of the wide spectrum P2 receptor antagonist PPADS, the selective P2X7 receptor antagonist Brilliant Blue G (BBG), the P2X1 receptor antagonist (4,4′,4″,4-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid, octasodium salt (NF449) and (8,8′-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonic acid, hexasodium salt (NF023), the P2Y12 receptor antagonist (2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester (MRS2395), the selective P2Y1 receptor agonist ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS2365), the P2Y2/P2Y4 agonist uridine-5′-triphosphate (UTP), and the P2Y4/P2Y6 agonist uridine-5′-diphosphate (UDP) were examined on mechanical allodynia in the Seltzer model of neuropathic pain, on acute thermal nociception, and on the inflammatory pain and oedema induced by complete Freund's adjuvant (CFA). Key results: MRS2365, MRS2395 and UTP, but not the other compounds, significantly alleviated mechanical allodynia in the neuropathic pain model, with the following rank order of minimal effective dose (mED) values: MRS2365 > MRS2395 > UTP. All compounds had a dose-dependent analgesic action in acute pain except BBG, which elicited hyperalgesia at a single dose. The rank order of mED values in acute pain was the following: MRS2365 > MRS2395 > NF449 > NF023 > UDP = UTP > PPADS. MRS2365 and MRS2395 had a profound, while BBG had a mild effect on inflammatory pain, with a following rank order of mED values: MRS2395 > MRS2365 > BBG. None of the tested

  6. WOX4 and WOX14 act downstream of the PXY receptor kinase to regulate plant vascular proliferation independently of any role in vascular organisation

    PubMed Central

    Etchells, J. Peter; Provost, Claire M.; Mishra, Laxmi; Turner, Simon R.

    2013-01-01

    In plants, the cambium and procambium are meristems from which vascular tissue is derived. In contrast to most plant cells, stem cells within these tissues are thin and extremely long. They are particularly unusual as they divide down their long axis in a highly ordered manner, parallel to the tangential axis of the stem. CLAVATA3-LIKE/ESR-RELATED 41 (CLE41) and PHLOEM INTERCALATED WITH XYLEM (PXY) are a multifunctional ligand-receptor pair that regulate vascular cell division, vascular organisation and xylem differentiation in vascular tissue. A transcription factor gene, WUSCHEL HOMEOBOX RELATED 4 (WOX4) has been shown to act downstream of PXY. Here we show that WOX4 acts redundantly with WOX14 in the regulation of vascular cell division, but that these genes have no function in regulating vascular organisation. Furthermore, we identify an interaction between PXY and the receptor kinase ERECTA (ER) that affects the organisation of the vascular tissue but not the rate of cell division, suggesting that cell division and vascular organisation are genetically separable. Our observations also support a model whereby tissue organisation and cell division are integrated via PXY and ER signalling, which together coordinate development of different cell types that are essential for normal stem formation. PMID:23578929

  7. A liver X receptor (LXR)-{beta} alternative splicing variant (LXRBSV) acts as an RNA co-activator of LXR-{beta}

    SciTech Connect

    Hashimoto, Koshi; Ishida, Emi; Matsumoto, Shunichi; Shibusawa, Nobuyuki; Okada, Shuichi; Monden, Tsuyoshi; Satoh, Tetsurou; Yamada, Masanobu; Mori, Masatomo

    2009-12-25

    We report the isolation and functional characterization of a novel transcriptional co-activator, termed LXRBSV. LXRBSV is an alternative splicing variant of liver X receptor (LXR)-{beta} LXRBSV has an intronic sequence between exons 2 and 3 in the mouse LXR-{beta} gene. The LXRBSV gene is expressed in various tissues including the liver and brain. We sub-cloned LXRBSV into pSG5, a mammalian expression vector, and LXRBSV in pSG5 augmented human Sterol Response Element Binding Protein (SREBP)-1c promoter activity in HepG2 cells in a ligand (TO901317) dependent manner. The transactivation mediated by LXRBSV is selective for LXR-{beta}. The LXRBSV protein was deduced to be 64 amino acids in length; however, a GAL4-LXRBSV fusion protein was not able to induce transactivation. Serial deletion constructs of LXRBSV demonstrated that the intronic sequence inserted in LXRBSV is required for its transactivation activity. An ATG mutant of LXRBSV was able to induce transactivation as wild type. Furthermore, LXRBSV functions in the presence of cycloheximide. Taken together, we have concluded that LXRBSV acts as an RNA transcript not as a protein. In the current study, we have demonstrated for the first time that an alternative splicing variant of a nuclear receptor acts as an RNA co-activator.

  8. The Use of Transdermal Buprenorphine in Complex Regional Pain Syndrome: A Report of Two Cases.

    PubMed

    Onofrio, Sarah; Vartan, Christine M; Nazario, Mitchell; DiScala, Sandra; Cuevas-Trisan, Ramon; Melendez-Benabe, John

    2016-06-01

    Management of complex regional pain syndrome (CRPS) can be challenging. Various pharmacological approaches have produced mixed results. Buprenorphine activates mu-opioid receptors and antagonizes kappa and delta receptors, acts at N-methyl-d-aspartate (NMDA) receptor, and is an orphan-related ligand-1 receptor agonist. It is available in transdermal patches that last for up to 7 days. This report describes two patients with refractory CRPS who were treated with transdermal buprenorphine. The patients experienced approximately 50% reduction in pain intensity scores. Application site rash that occurred was managed with topical steroid spray used before applying the patch. PMID:27172230

  9. Early cerebral activities of the environmental estrogen bisphenol A appear to act via the somatostatin receptor subtype sst(2).

    PubMed Central

    Facciolo, Rosa Maria; Alò, Raffaella; Madeo, Maria; Canonaco, Marcello; Dessì-Fulgheri, Francesco

    2002-01-01

    Recently, considerable interest has been aroused by the specific actions of bisphenol A (BPA). The present investigation represents a first study dealing with the interaction of BPA with the biologically more active somatostatin receptor subtype (sst(2)) in the rat limbic circuit. After treating pregnant female Sprague-Dawley rats with two doses (400 microg/kg/day; 40 microg/kg/day) of BPA, the binding activity of the above receptor subtype was evaluated in some limbic regions of the offspring. The higher dose proved to be the more effective one, as demonstrated by the elevated affinity of sst(2) with its specific radioligand, [(125)I]-Tyr(0)somatostatin-14. The most dramatic effects of BPA on sst(2) levels occurred at the low-affinity states of such a subtype in some telencephalic limbic areas of postnatal rats (10 days of age; postnatal day [PND] 10). These included lower (p < 0.05) sst(2) levels in the gyrus dentate of the hippocampus and basomedial nucleus of the amygdala; significantly higher (p < 0.01) levels were observed only for the high-affinity states of the periventricular nucleus of the hypothalamus. A similar trend was maintained in PND 23 rats with the exception of much lower levels of the high-affinity sst(2) receptor subtype in the amygdala nucleus and ventromedial hypothalamic nucleus. However, greater changes produced by this environmental estrogen were reported when the binding activity of sst(2) was checked in the presence of the two more important selective agonists (zolpidem and Ro 15-4513) specific for the alpha-containing Gamma-aminobutyric acid (GABA) type A receptor complex. In this case, an even greater potentiating effect (p < 0.001) was mainly obtained for the low-affinity sst(2) receptor subtype in PND 10 animals, with the exception of the high-affinity type in the ventromedial hypothalamic nucleus and gyrus dentate. These results support the contention that an sst(2) subtype alpha-containing GABA type A receptor system might

  10. Angiotensin II centrally induces frequent detrusor contractility of the bladder by acting on brain angiotensin II type 1 receptors in rats

    PubMed Central

    Kawamoto, Bunya; Shimizu, Shogo; Shimizu, Takahiro; Higashi, Youichirou; Honda, Masashi; Sejima, Takehiro; Saito, Motoaki; Takenaka, Atsushi

    2016-01-01

    Angiotensin (Ang) II plays an important role in the brain as a neurotransmitter and is involved in psychological stress reactions, for example through activation of the sympatho-adrenomedullary system. We investigated the effects of centrally administered Ang II on the micturition reflex, which is potentially affected by the sympatho-adrenomedullary system, and brain Ang II receptors in urethane-anesthetized (1.0 g/kg, intraperitoneally) male rats. Central administration of Ang II (0.01, 0.02, and 0.07 nmol per rat, intracerebroventricularly, icv) but not vehicle rapidly and dose-dependently decreased the urinary bladder intercontraction interval, without altering the bladder detrusor pressure. Central administration of antagonists of Ang II type 1 but not type 2 receptors inhibited the Ang II-induced shortening of intercontraction intervals. Administration of the highest dose of Ang II (0.07 nmol per rat, icv) but not lower doses (0.01 and 0.02 nmol per rat, icv) elevated the plasma concentration of adrenaline. Bilateral adrenalectomy reduced Ang II-induced elevation in adrenaline, but had no effect on the Ang II-induced shortening of the intercontraction interval. These data suggest that central administration of Ang II increases urinary frequency by acting on brain Ang II type 1 receptors, independent of activation of the sympatho-adrenomedullary system. PMID:26908391

  11. Glutamate Receptor Antagonists as Fast-Acting Therapeutic Alternatives for the Treatment of Depression: Ketamine and Other Compounds

    PubMed Central

    Niciu, Mark J.; Henter, Ioline D.; Luckenbaugh, David A.; Zarate, Carlos A.; Charney, Dennis S.

    2014-01-01

    The N-methyl-d-aspartate (NMDA) receptor antagonist ketamine has rapid and potent antidepressant effects in treatment-resistant major depressive disorder and bipolar depression. These effects are in direct contrast to the more modest effects seen after weeks of treatment with classic monoaminergic antidepressants. Numerous open-label and case studies similarly validate ketamine’s antidepressant properties. These clinical findings have been reverse-translated into preclinical models in an effort to elucidate ketamine’s antidepressant mechanism of action, and three important targets have been identified: mammalian target of rapamycin (mTOR), eukaryotic elongation factor 2 (eEF2), and glycogen synthase kinase-3 (GSK-3). Current clinical and preclinical research is focused on (a) prolonging/maintaining ketamine’s antidepressant effects, (b) developing more selective NMDA receptor antagonists free of ketamine’s adverse effects, and (c) identifying predictor, mediator/moderator, and treatment response biomarkers of ketamine’s antidepressant effects. PMID:24392693

  12. Vascular Endothelial Growth Factor Acts Primarily via Platelet-Derived Growth Factor Receptor α to Promote Proliferative Vitreoretinopathy

    PubMed Central

    Pennock, Steven; Haddock, Luis J.; Mukai, Shizuo; Kazlauskas, Andrius

    2015-01-01

    Proliferative vitreoretinopathy (PVR) is a nonneovascular blinding disease and the leading cause for failure in surgical repair of rhegmatogenous retinal detachments. Once formed, PVR is difficult to treat. Hence, there is an acute interest in developing approaches to prevent PVR. Of the many growth factors and cytokines that accumulate in vitreous as PVR develops, neutralizing vascular endothelial growth factor (VEGF) A has recently been found to prevent PVR in at least one animal model. The goal of this study was to test if Food and Drug Administration–approved agents could protect the eye from PVR in multiple animal models and to further investigate the underlying mechanisms. Neutralizing VEGF with aflibercept (VEGF Trap-Eye) safely and effectively protected rabbits from PVR in multiple models of disease. Furthermore, aflibercept reduced the bioactivity of both experimental and clinical PVR vitreous. Finally, although VEGF could promote some PVR-associated cellular responses via VEGF receptors expressed on the retinal pigment epithelial cells that drive this disease, VEGF's major contribution to vitreal bioactivity occurred via platelet-derived growth factor receptor α. Thus, VEGF promotes PVR by a noncanonical ability to engage platelet-derived growth factor receptor α. These findings indicate that VEGF contributes to nonangiogenic diseases and that anti–VEGF-based therapies may be effective on a wider spectrum of diseases than previously appreciated. PMID:25261788

  13. Cardiovascular protection and antioxidant activity of the extracts from the mycelia of Cordyceps sinensis act partially via adenosine receptors.

    PubMed

    Yan, Xiao-Feng; Zhang, Zhong-Miao; Yao, Hong-Yi; Guan, Yan; Zhu, Jian-Ping; Zhang, Lin-Hui; Jia, Yong-Liang; Wang, Ru-Wei

    2013-11-01

    Mycelia of cultured Cordyceps sinensis (CS) is one of the most common substitutes for natural CS and was approved for arrhythmia in China. However, the role of CS in ameliorating injury during ischemia-reperfusion (I/R) is still unclear. We examined effects of extracts from CS on I/R and investigated the possible mechanisms. Post-ischemic coronary perfusion pressure, ventricular function, and coronary flow were measured using the Langendorff mouse heart model. Oxidative stress of cardiac homogenates was performed using an ELISA. Our results indicate that CS affords cardioprotection possibly through enhanced adenosine receptor activation. Cardioprotection was demonstrated by reduced post-ischemic diastolic dysfunction and improved recovery of pressure development and coronary flow. Treatment with CS largely abrogates oxidative stress and damage in glucose- or pyruvate-perfused hearts. Importantly, observed reductions in oxidative stress [glutathione disulfide (GSSG)]/[GSSG + glutathione] and [malondialdehyde (MDA)]/[superoxide dismutase + MDA] ratios as well as the resultant damage upon CS treatment correlate with functional markers of post-ischemic myocardial outcome. These effects of CS were partially blocked by 8-ρ-sulfophenyltheophylline, an adenosine receptor antagonist. Our results demonstrate a suppressive role of CS in ischemic contracture. Meanwhile, the results also suggest pre-ischemic adenosine receptor activation may be involved in reducing contracture in hearts pretreated with CS. PMID:23192916

  14. GGNBP2 acts as a tumor suppressor by inhibiting estrogen receptor α activity in breast cancer cells.

    PubMed

    Lan, Zi-Jian; Hu, YunHui; Zhang, Sheng; Li, Xian; Zhou, Huaxin; Ding, Jixiang; Klinge, Carolyn M; Radde, Brandie N; Cooney, Austin J; Zhang, Jin; Lei, Zhenmin

    2016-07-01

    Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E2)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis. PMID:27357812

  15. Preliminary Investigation into a Potential Role for Myostatin and Its Receptor (ActRIIB) in Lean and Obese Horses and Ponies

    PubMed Central

    Morrison, Philippa K.; Bing, Chen; Harris, Patricia A.; Maltin, Charlotte A.; Grove-White, Dai; Argo, Caroline McG.

    2014-01-01

    Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obese animals to explore putative associations between these factors and obesity in horses and ponies. Myostatin mRNA expression was increased while ActRIIB mRNA was decreased in skeletal muscles of obese animals but these differences were absent at the protein level. Myostatin mRNA was increased in crest fat of obese animals but neither myostatin nor ActRIIB proteins were detected in this tissue. Mean circulating myostatin concentrations were significantly higher in obese than in lean groups; 4.98 ng/ml (±2.71) and 9.00 ng/ml (±2.04) for the lean and obese groups, respectively. In addition, there was a significant positive association between these levels and myostatin gene expression in skeletal muscles (average R2 = 0.58; p<0.05). Together, these results provide further basis for the speculation that myostatin and its receptor may play a role in obesity in horses and ponies. PMID:25390640

  16. Preliminary investigation into a potential role for myostatin and its receptor (ActRIIB) in lean and obese horses and ponies.

    PubMed

    Morrison, Philippa K; Bing, Chen; Harris, Patricia A; Maltin, Charlotte A; Grove-White, Dai; Argo, Caroline McG

    2014-01-01

    Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obese animals to explore putative associations between these factors and obesity in horses and ponies. Myostatin mRNA expression was increased while ActRIIB mRNA was decreased in skeletal muscles of obese animals but these differences were absent at the protein level. Myostatin mRNA was increased in crest fat of obese animals but neither myostatin nor ActRIIB proteins were detected in this tissue. Mean circulating myostatin concentrations were significantly higher in obese than in lean groups; 4.98 ng/ml (±2.71) and 9.00 ng/ml (±2.04) for the lean and obese groups, respectively. In addition, there was a significant positive association between these levels and myostatin gene expression in skeletal muscles (average R2 = 0.58; p<0.05). Together, these results provide further basis for the speculation that myostatin and its receptor may play a role in obesity in horses and ponies. PMID:25390640

  17. In silico identification and pharmacological evaluation of novel endocrine disrupting chemicals that act via the ligand-binding domain of the estrogen receptor α.

    PubMed

    McRobb, Fiona M; Kufareva, Irina; Abagyan, Ruben

    2014-09-01

    Endocrine disrupting chemicals (EDCs) pose a significant threat to human health, society, and the environment. Many EDCs elicit their toxic effects through nuclear hormone receptors, like the estrogen receptor α (ERα). In silico models can be used to prioritize chemicals for toxicological evaluation to reduce the amount of costly pharmacological testing and enable early alerts for newly designed compounds. However, many of the current computational models are overly dependent on the chemistry of known modulators and perform poorly for novel chemical scaffolds. Herein we describe the development of computational, three-dimensional multi-conformational pocket-field docking, and chemical-field docking models for the identification of novel EDCs that act via the ligand-binding domain of ERα. These models were highly accurate in the retrospective task of distinguishing known high-affinity ERα modulators from inactive or decoy molecules, with minimal training. To illustrate the utility of the models in prospective in silico compound screening, we screened a database of over 6000 environmental chemicals and evaluated the 24 top-ranked hits in an ERα transcriptional activation assay and a differential scanning fluorimetry-based ERα binding assay. Promisingly, six chemicals displayed ERα agonist activity (32nM-3.98μM) and two chemicals had moderately stabilizing effects on ERα. Two newly identified active compounds were chemically related β-adrenergic receptor (βAR) agonists, dobutamine, and ractopamine (a feed additive that promotes leanness in cattle and poultry), which are the first βAR agonists identified as activators of ERα-mediated gene transcription. This approach can be applied to other receptors implicated in endocrine disruption. PMID:24928891

  18. In Silico Identification and Pharmacological Evaluation of Novel Endocrine Disrupting Chemicals That Act via the Ligand-Binding Domain of the Estrogen Receptor α

    PubMed Central

    Kufareva, Irina; Abagyan, Ruben

    2014-01-01

    Endocrine disrupting chemicals (EDCs) pose a significant threat to human health, society, and the environment. Many EDCs elicit their toxic effects through nuclear hormone receptors, like the estrogen receptor α (ERα). In silico models can be used to prioritize chemicals for toxicological evaluation to reduce the amount of costly pharmacological testing and enable early alerts for newly designed compounds. However, many of the current computational models are overly dependent on the chemistry of known modulators and perform poorly for novel chemical scaffolds. Herein we describe the development of computational, three-dimensional multi-conformational pocket-field docking, and chemical-field docking models for the identification of novel EDCs that act via the ligand-binding domain of ERα. These models were highly accurate in the retrospective task of distinguishing known high-affinity ERα modulators from inactive or decoy molecules, with minimal training. To illustrate the utility of the models in prospective in silico compound screening, we screened a database of over 6000 environmental chemicals and evaluated the 24 top-ranked hits in an ERα transcriptional activation assay and a differential scanning fluorimetry-based ERα binding assay. Promisingly, six chemicals displayed ERα agonist activity (32nM–3.98μM) and two chemicals had moderately stabilizing effects on ERα. Two newly identified active compounds were chemically related β-adrenergic receptor (βAR) agonists, dobutamine, and ractopamine (a feed additive that promotes leanness in cattle and poultry), which are the first βAR agonists identified as activators of ERα-mediated gene transcription. This approach can be applied to other receptors implicated in endocrine disruption. PMID:24928891

  19. Gabapentin and the neurokinin(1) receptor antagonist CI-1021 act synergistically in two rat models of neuropathic pain.

    PubMed

    Field, Mark J; Gonzalez, M Isabel; Tallarida, Ronald J; Singh, Lakhbir

    2002-11-01

    The present study examines the effect of combinations of gabapentin (Neurontin) and a selective neurokinin (NK)(1) receptor antagonist, 1-(1H-indol-3-ylmethyl)-1-methyl-2-oxo-2-[(1-phenylethyl)amino]ethyl]-2-benzofuranylmethyl ester (CI-1021), in two models of neuropathic pain. Dose responses to both gabapentin and CI-1021 were performed against static allodynia induced in the streptozocin and chronic constriction injury (CCI) models. Theoretical additive lines were calculated from these data. Dose responses to various fixed dose ratios of a gabapentin/CI-1021 combination were then examined in both models. In the streptozocin model, administration of gabapentin/CI-1021 combinations at fixed dose ratios of 1:1 and 60:1 resulted in an additive effect with dose response similar to the theoretical additive line. However, a synergistic interaction was seen after fixed dose ratios of 10:1, 20:1, and 40:1 with static allodynia completely blocked and the dose responses shifted approximately 8-, 30-, and 10-fold leftward, respectively, from the theoretical additive values. In the CCI model, after fixed dose ratios of 5:1 and 20:1, combinations of gabapentin and CI-1021 produced an additive response. At the fixed dose ratio of 10:1 static allodynia was completely blocked with an approximate 10-fold leftward shift of the dose response from the theoretical additive value, indicating synergy. The combination of gabapentin with a structurally unrelated NK(1) receptor antagonist, (2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP-99,994), also produced synergy, at a fixed dose ratio of 20:1. This ratio completely blocked streptozocin-induced static allodynia and was approximately shifted leftward 5-fold from the theoretical additive value. These data suggest a synergistic interaction between gabapentin and NK(1) receptor antagonists in animal models of neuropathic pain. PMID:12388658

  20. Cyclohexanol analogues are positive modulators of GABA(A) receptor currents and act as general anaesthetics in vivo.

    PubMed

    Hall, Adam C; Griffith, Theanne N; Tsikolia, Maia; Kotey, Francesca O; Gill, Nikhila; Humbert, Danielle J; Watt, Erin E; Yermolina, Yuliya A; Goel, Shikha; El-Ghendy, Bahaa; Hall, C Dennis

    2011-09-30

    GABA(A) receptors meet all the pharmacological criteria required to be considered important general anaesthetic targets. In the following study, the modulatory effects of various commercially available and novel cyclohexanols were investigated on recombinant human γ-aminobutyric acid (GABA(A), α(1)β(2)γ(2s)) receptors expressed in Xenopus oocytes, and compared to the modulatory effects on GABA currents observed with exposures to the intravenous anaesthetic agent, propofol. Submaximal EC(20) GABA currents were typically enhanced by co-applications of 3-300 μM cyclohexanols. For instance, at 30 μM 2,6-diisopropylcyclohexanol (a novel compound) GABA responses were increased ~3-fold (although similar enhancements were achieved at 3 μM propofol). As regards rank order for modulation by the cyclohexanol analogues at 30 μM, the % enhancements for 2,6-dimethylcyclohexanol~2,6-diethylcyclohexanol~2,6-diisopropylcyclohexanol~2,6-di-sec-butylcyclohexanol ≫2,6-di-tert-butylcyclohexanol~4-tert-butylcyclohexanol>cyclohexanol~cyclopentanol~2-methylcyclohexanol. We further tested the potencies of the cyclohexanol analogues as general anaesthetics using a tadpole in vivo assay. Both 2,6-diisopropylcyclohexanol and 2,6-dimethylcyclohexanol were effective as anaesthetics with EC(50)s of 14.0 μM and 13.1 μM respectively, while other cyclohexanols with bulkier side chains were less potent. In conclusion, our data indicate that cyclohexanols are both positive modulators of GABA(A) receptors currents and anaesthetics. The positioning and size of the alkyl groups at the 2 and 6 positions on the cyclohexanol ring were critical determinants of activity. PMID:21658385

  1. β-arrestin-2 regulates NMDA receptor function in spinal lamina II neurons and duration of persistent pain.

    PubMed

    Chen, Gang; Xie, Rou-Gang; Gao, Yong-Jing; Xu, Zhen-Zhong; Zhao, Lin-Xia; Bang, Sangsu; Berta, Temugin; Park, Chul-Kyu; Lay, Mark; Chen, Wei; Ji, Ru-Rong

    2016-01-01

    Mechanisms of acute pain transition to chronic pain are not fully understood. Here we demonstrate an active role of β-arrestin 2 (Arrb2) in regulating spinal cord NMDA receptor (NMDAR) function and the duration of pain. Intrathecal injection of the mu-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin produces paradoxical behavioural responses: early-phase analgesia and late-phase mechanical allodynia which requires NMDAR; both phases are prolonged in Arrb2 knockout (KO) mice. Spinal administration of NMDA induces GluN2B-dependent mechanical allodynia, which is prolonged in Arrb2-KO mice and conditional KO mice lacking Arrb2 in presynaptic terminals expressing Nav1.8. Loss of Arrb2 also results in prolongation of inflammatory pain and neuropathic pain and enhancement of GluN2B-mediated NMDA currents in spinal lamina IIo not lamina I neurons. Finally, spinal over-expression of Arrb2 reverses chronic neuropathic pain after nerve injury. Thus, spinal Arrb2 may serve as an intracellular gate for acute to chronic pain transition via desensitization of NMDAR. PMID:27538456

  2. Modulation of Ca2+ channels by opioid receptor antagonists in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

    PubMed

    Kai, Li; Wang, Zhong-Feng; Hu, De-Yao; Shi, Yu-Liang; Liu, Liang-Ming

    2002-10-01

    The effects of hemorrhagic shock on Ba currents ( ) via Ca channels and the regulation of the channels in the vascular hyporesponse stage of hemorrhagic shock by opioid receptor antagonists were examined by using the whole-cell recording of patch-clamp technique in mesenteric arterial smooth muscle cells of rats. The results showed that hemorrhagic shock induced an inhibition of Ca channels in the cells; 10 micro M of naloxone and 100 n of naltrindole, nor-binaltorphimine, and beta-funaltrexamine increased the in the cells of rats in shock. After inhibition of protein kinase C by using 1-(5-isoquindinesulfonyl)-2-methylpiperazine via electrodes, the enhancement of by the antagonists was not observed. These results suggested that the inhibition of Ca channel induced by hemorrhagic shock was mediated by delta-, kappa-, and mu -opioid receptors in the cells and may be partly responsible for vascular hyporesponse. The enhancement of was mediated by activation of protein kinase C and may be responsible for the antagonist-caused improvement in the response of resistance arteries to vasoactive stimulants at the decompensatory stage of hemorrhagic shock. PMID:12352325

  3. Pharmacological characterization of FE 202158, a novel, potent, selective, and short-acting peptidic vasopressin V1a receptor full agonist for the treatment of vasodilatory hypotension.

    PubMed

    Laporte, Régent; Kohan, Arash; Heitzmann, Joshua; Wisniewska, Halina; Toy, Jeannine; La, Erin; Tariga, Hiroe; Alagarsamy, Sudarkodi; Ly, Brian; Dykert, John; Qi, Steve; Wisniewski, Kazimierz; Galyean, Robert; Croston, Glenn; Schteingart, Claudio D; Rivière, Pierre J-M

    2011-06-01

    FE 202158, ([Phe(2),Ile(3),Hgn(4),Orn(iPr)(8)]vasopressin, where Hgn is homoglutamine and iPr is isopropyl), a peptidic analog of the vasoconstrictor hormone [Arg(8)]vasopressin (AVP), was designed to be a potent, selective, and short-acting vasopressin type 1a receptor (V(1a)R) agonist. In functional reporter gene assays, FE 202158 was a potent and selective human V(1a)R agonist [EC(50) = 2.4 nM; selectivity ratio of 1:142:1107:440 versus human vasopressin type 1b receptor, vasopressin type 2 receptor (V(2)R), and oxytocin receptor, respectively] contrasting with AVP's lack of selectivity, especially versus the V(2)R (selectivity ratio of 1:18:0.2:92; human V(1a)R EC(50) = 0.24 nM). This activity and selectivity profile was confirmed in radioligand binding assays. FE 202158 was a potent vasoconstrictor in the isolated rat common iliac artery ex vivo (EC(50) = 3.6 nM versus 0.8 nM for AVP) and reduced rat ear skin blood flow after intravenous infusion in vivo (ED(50) = 4.0 versus 3.4 pmol/kg/min for AVP). The duration of its vasopressor effect by intravenous bolus in rats was as short as AVP at submaximally effective doses. FE 202158 had no V(2)R-mediated antidiuretic activity in rats by intravenous infusion at its ED(50) for reduction of ear skin blood flow, in contrast with the pronounced antidiuretic effect of AVP. Thus, FE 202158 seems suitable for treatment of conditions where V(1a)R activity is desirable but V(2)R activity is potentially deleterious, such as vasodilatory hypotension in septic shock. In addition to the desirable selectivity profile, its short-acting nature should allow dose titration with rapid onset and offset of action to optimize vasoconstriction efficacy and safety. PMID:21411496

  4. Fatigue-induced Orosomucoid 1 Acts on C-C Chemokine Receptor Type 5 to Enhance Muscle Endurance

    PubMed Central

    Lei, Hong; Sun, Yang; Luo, Zhumin; Yourek, Gregory; Gui, Huan; Yang, Yili; Su, Ding-Feng; Liu, Xia

    2016-01-01

    Understanding and managing fatigue is a significant challenge in clinic and society. In attempting to explore how the body responds to and regulates fatigue, we found in rodent fatigue models that orosomucoid 1 (ORM1) was significantly increased in multiple tissues, including blood and muscle. Interestingly, administration of exogenous ORM1 increased muscle glycogen and enhanced muscle endurance, whereas ORM1 deficiency resulted in a significant decrease of muscle endurance both in vivo and in vitro, which could largely be restored by exogenous ORM1. Further studies demonstrated that ORM1 can bind to C-C chemokine receptor type 5 (CCR5) on muscle cells and deletion of the receptor abolished the effect of ORM1. Thus, fatigue upregulates the level of ORM1, which in turn functions as an anti-fatigue protein to enhance muscle endurance via the CCR5 pathway. Modulation of the level of ORM1 and CCR5 signaling could be a novel strategy for the management of fatigue. PMID:26740279

  5. VIP, CRF, and PACAP Act at Distinct Receptors to Elicit Different cAMP/PKA Dynamics in the Neocortex

    PubMed Central

    Hu, Emilie; Demmou, Lynda; Cauli, Bruno; Gallopin, Thierry; Geoffroy, Hélène; Harris-Warrick, Ronald M.; Paupardin-Tritsch, Danièle; Lambolez, Bertrand; Vincent, Pierre

    2011-01-01

    The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase–activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription–polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects. PMID:20699230

  6. A Compartment Model of VEGF Distribution in Humans in the Presence of Soluble VEGF Receptor-1 Acting as a Ligand Trap

    PubMed Central

    Wu, Florence T. H.; Stefanini, Marianne O.; Mac Gabhann, Feilim; Popel, Aleksander S.

    2009-01-01

    Vascular endothelial growth factor (VEGF), through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis – new capillary growth from existing microvasculature – at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1) – a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains – has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis–dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (vascular permeability, lymphatic drainage); and molecularly-detailed binding interactions between the ligand isoforms VEGF121 and VEGF165, signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1), as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 – acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization

  7. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    SciTech Connect

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  8. Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

    SciTech Connect

    Notcovich, Cintia; Diez, Federico; Tubio, Maria Rosario; Baldi, Alberto; Kazanietz, Marcelo G.; Davio, Carlos; Shayo, Carina

    2010-02-01

    It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G{sub 11}-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP {beta}2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or {beta}2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [{sup 3}H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.

  9. Synaptic actions of neuropeptide FF in the rat parabrachial nucleus: interactions with opioid receptors.

    PubMed

    Chen, X; Zidichouski, J A; Harris, K H; Jhamandas, J H

    2000-08-01

    The pontine parabrachial nucleus (PBN) receives both opioid and Neuropeptide FF (NPFF) projections from the lower brain stem and/or the spinal cord. Because of this anatomical convergence and previous evidence that NPFF displays both pro- and anti-opioid activities, this study examined the synaptic effects of NPFF in the PBN and the mechanisms underlying these effects using an in vitro brain slice preparation and the nystatin-perforated patch-clamp recording technique. Under voltage-clamp conditions, NPFF reversibly reduced the evoked excitatory postsynaptic currents (EPSCs) in a dose-dependent fashion. This effect was not accompanied by apparent changes in the holding current, the current-voltage relationship or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced inward currents in the PBN cells. When a paired-pulse protocol was used, NPFF increased the ratio of these synaptic currents. Analysis of miniature EPSCs showed that NPFF caused a rightward shift in the frequency-distribution curve, whereas the amplitude-distribution curve remained unchanged. Collectively, these experiments indicate that NPFF reduces the evoked EPSCs through a presynaptic mechanism of action. The synaptic effects induced by NPFF (5 microM) could not be blocked by the specific mu-opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (1 microM), but application of delta-opioid receptor antagonist Tyr-Tic-Phe-Phe (5 microM) almost completely prevented effects of NPFF. Moreover, the delta-opioid receptor agonist, Deltorphin (1 microM), mimicked the effects as NPFF and also occluded NPFF's actions on synaptic currents. These results indicate that NPFF modulates excitatory synaptic transmission in the PBN through an interaction with presynaptic delta-opioid receptors. These observations provide a cellular basis for NPFF enhancement of the antinociceptive effects consequent to central activation of delta-opioid receptors. PMID:10938301

  10. Oxytocin (OT) and arginine-vasopressin (AVP) act on OT receptors and not AVP V1a receptors to enhance social recognition in adult Syrian hamsters (Mesocricetus auratus).

    PubMed

    Song, Zhimin; Larkin, Tony E; Malley, Maureen O'; Albers, H Elliott

    2016-05-01

    Social recognition is a fundamental requirement for all forms of social relationships. A majority of studies investigating the neural mechanisms underlying social recognition in rodents have investigated relatively neutral social stimuli such as juveniles or ovariectomized females over short time intervals (e.g., 2h). The present study developed a new testing model to study social recognition among adult males using a potent social stimulus. Flank gland odors are used extensively in social communication in Syrian hamsters and convey important information such as dominance status. We found that the recognition of flank gland odors after a 3min exposure lasted for at least 24h, substantially longer than the recognition of other social cues in rats and mice. Intracerebroventricular injections of OT and AVP prolonged the recognition of flank gland odor for up to 48h. Selective OTR but not V1aR agonists, mimicked these enhancing effects of OT and AVP. Similarly, selective OTR but not V1aR antagonists blocked recognition of the odors after 20min. In contrast, the recognition of non-social stimuli was not blocked by either the OTR or the V1aR antagonists. Our findings suggest both OT and AVP enhance social recognition via acting on OTRs and not V1aRs and that the recognition enhancing effects of OT and AVP are limited to social stimuli. PMID:26975586

  11. Indole Glucocorticoid Receptor Antagonists Active in a Model of Dyslipidemia Act via a Unique Association with an Agonist Binding Site.

    PubMed

    Luz, John G; Carson, Matthew W; Condon, Bradley; Clawson, David; Pustilnik, Anna; Kohlman, Daniel T; Barr, Robert J; Bean, James S; Dill, M Joelle; Sindelar, Dana K; Maletic, Milan; Coghlan, Michael J

    2015-08-27

    To further elucidate the structural activity correlation of glucocorticoid receptor (GR) antagonism, the crystal structure of the GR ligand-binding domain (GR LBD) complex with a nonsteroidal antagonist, compound 8, was determined. This novel indole sulfonamide shows in vitro activity comparable to known GR antagonists such as mifepristone, and notably, this molecule lowers LDL (-74%) and raises HDL (+73%) in a hamster model of dyslipidemia. This is the first reported crystal structure of the GR LBD bound to a nonsteroidal antagonist, and this article provides additional elements for the design and pharmacology of clinically relevant nonsteroidal GR antagonists that may have greater selectivity and fewer side effects than their steroidal counterparts. PMID:26218343

  12. Application of the functionalized congener approach to dendrimer-based signaling agents acting through A(2A) adenosine receptors.

    PubMed

    Kim, Yoonkyung; Klutz, Athena M; Hechler, Béatrice; Gao, Zhan-Guo; Gachet, Christian; Jacobson, Kenneth A

    2009-03-01

    As a continued effort to develop multivalent ligands to enhance the pharmacological effects of monomeric drugs, DITC-APEC, a chemically reactive nucleoside A(2A) adenosine receptor (AR) agonist, was employed to derivatize the surface of third-generation (G3) polyamidoamine (PAMAM) dendrimers. The resulting conjugates carried multiple copies of the agonist attached through a thiourea linkage and differed in the number of attachments and in the presence of a fluorophore or additional surface modification. Computer modeling studies suggested that these DITC-APEC-loaded dendrimers extended the overall diameter of the previously reported PAMAM-CGS21680 dendrimer derivatives (Kim et al., Bioconjugate Chem 2008 19:406-411) by ca. 20 A, potentially increasing the conformational flexibility of the appended ligands to achieve optimal geometry for efficient binding at A(2A) ARs. Increased affinity and selectivity in binding in comparison to the CGS21680 conjugate were envisioned, due to the presence of an extended linker, i.e., a dithioureylenephenyl functionality. In vitro radioligand competition experiments showed effective binding of these PAMAM-DITC-APEC dendrimer conjugates at the human A(2A) and A(3) ARs with submicromolar K (i) values and selectivity in comparison to the human A(1) AR. Furthermore, these nucleoside-loaded dendrimers exhibited an A(2A) AR-mediated inhibitory effect on ADP-induced aggregation of human platelets. The present study demonstrates the potential of applying the functionalized congener concept to engineer dendrimer-based multivalent ligands for G protein-coupled receptors. PMID:18600474

  13. Chemotactic action of prostaglandin E2 on mouse mast cells acting via the PGE2 receptor 3

    PubMed Central

    Weller, Charlotte L.; Collington, Sarah J.; Hartnell, Adele; Conroy, Dolores M.; Kaise, Toshihiko; Barker, Jane E.; Wilson, Mark S.; Taylor, Graham W.; Jose, Peter J.; Williams, Timothy J.

    2007-01-01

    Mast cells are long-lived cells that are principally recognized for their effector function in helminth infections and allergic reactions. These cells are derived from pluripotential hematopoietic stem cells in the bone marrow that give rise to committed mast cell progenitors in the blood and are recruited to tissues, where they mature. Little is known about the chemotactic signals responsible for recruitment of progenitors and localization of mature mast cells. A mouse model was set up to identify possible mast cell progenitor chemoattractants produced during repeated allergen challenge in vivo. After the final challenge, the nasal mucosa was removed to produce conditioned medium, which was tested in chemotaxis assays against 2-wk murine bone marrow-derived c-kit+ mast cells (BMMC). A single peak of chemotactic activity was seen on reverse-phase HPLC with a retention time and electrospray mass spectrum consistent with prostaglandin E2 (PGE2). This lipid was found to be a highly potent chemoattractant for immature (2-wk) and also mature (10-wk) BMMC in vitro. Fluorescently labeled 2-wk c-kit+ BMMC, when injected intravenously, accumulated in response to intradermally injected PGE2. Analysis using TaqMan showed mRNA expression of the PGE2 receptors 3 (EP3) and 4 (EP4) on 2- and 10-wk BMMC. Chemotaxis induced by PGE2 was mimicked by EP3 agonists, blocked by an EP3 receptor antagonist, and partially inhibited by a MAPKK inhibitor. These results show an unexpected function for PGE2 in the chemotaxis of mast cells. PMID:17606905

  14. Dopamine-related drugs act presynaptically to potentiate GABA(A) receptor currents in VTA dopamine neurons.

    PubMed

    Michaeli, Avner; Yaka, Rami

    2011-01-01

    Electrical activity of ventral tegmental area (VTA) dopamine (DA) neurons is immediately inhibited following in vivo administration of cocaine and other DA-related drugs. While various forms of synaptic modulation were demonstrated in the VTA following exposure to DA-related drugs, comprehensive understanding of their ability to inhibit the activity of DA neurons, however, is still lacking. In this study, using whole-cell patch-clamp recordings from rat brain slices, a novel form of synaptic modulation induced by DA-related drugs was isolated. DA exposure was shown to cause potentiation of γ-amino-butyric acid (GABA) receptor type A (GABA(A)R)-mediated evoked inhibitory postsynaptic currents (eIPSCs), recorded from VTA DA neurons, under conditions of potassium channels blockade. The potentiation of these eIPSCs lasted for more than twenty minutes, could be mimicked by activation of D2-like but not D1-like DA receptors, and was accompanied by an increase in the frequency of GABA(A)R-mediated spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Furthermore, exposure to inhibitors of DA transporter (DAT) led to potentiation of GABA(A) currents in a manner similar to the DA-mediated potentiation. Finally, a prolonged presence of l-NAME, an inhibitor of nitric-oxide (NO) signaling was found to conceal the potentiation of GABA(A) currents induced by the DA-related drugs. Taken together, this study demonstrates a new modulatory form of VTA GABA(A) neurotransmission mediated by DA-related drugs. These results also suggest better understanding of the initial inhibitory action of DA-related drugs on the activity of DA neurons in the VTA. PMID:21527263

  15. Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress.

    PubMed

    Kaster, Manuella P; Machado, Nuno J; Silva, Henrique B; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E; Rodrigues, Ana Lúcia S; Porciúncula, Lisiane O; Chen, Jiang Fan; Tomé, Ângelo R; Agostinho, Paula; Canas, Paula M; Cunha, Rodrigo A

    2015-06-23

    The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function. PMID:26056314

  16. Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

    PubMed Central

    Kaster, Manuella P.; Machado, Nuno J.; Silva, Henrique B.; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E.; Rodrigues, Ana Lúcia S.; Porciúncula, Lisiane O.; Chen, Jiang Fan; Tomé, Ângelo R.; Agostinho, Paula; Canas, Paula M.; Cunha, Rodrigo A.

    2015-01-01

    The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function. PMID:26056314

  17. Transfer of SAR information from hypotensive indazole to indole derivatives acting at α-adrenergic receptors: In vitro and in vivo studies.

    PubMed

    Sączewski, Jaroslaw; Hudson, Alan; Scheinin, Mika; Wasilewska, Aleksandra; Sączewski, Franciszek; Rybczyńska, Apolonia; Ferdousi, Mehnaz; Laurila, Jonne M; Boblewski, Konrad; Lehmann, Artur; Watts, Helena; Ma, Daqing

    2016-06-10

    In a search for novel antihypertensive drugs we applied scaffold hopping from the previously described α1-adrenergic receptor antagonists, 1-[(imidazolin-2-yl)methyl]indazoles. The aim was to investigate whether the α-adrenergic properties of the indazole core were transferable to the indole core. The newly obtained 1-[(imidazolin-2-yl)methyl]indole analogues were screened in vitro for their binding affinities for α1-and α2-adrenoceptors, which allowed the identification of the target-based SAR transfer (T_SAR transfer) as well as structure-based SAR transfer (S_SAR transfer) events. However, when screened in vivo with use of anaesthetized male Wistar rats, the new indole ligands showed a different hemodynamic profile than expected. Instead of the immediate hypotensive effect characteristic of peripheral vasodilatator α1 blockers, a biphasic effect was observed, reminiscent of clonidine-like centrally acting antihypertensive agents. This was supported by subsequent in vitro functional studies in [(35)S]GTPγS binding assay, where the indole analogues displayed partial agonist properties at α2-adrenergic receptors. Since no correlation was found between the in vitro binding to α-adrenoceptors and the in vivo hemodynamic effects of the two series of indazole and indole bioisosteric compounds, in a search for new imidazoline-containing adrenergic drugs, the structure-based SAR transfer information obtained from in vitro binding studies should be treated with caution. PMID:27031216

  18. Juvenile Hormone-Receptor Complex Acts on Mcm4 and Mcm7 to Promote Polyploidy and Vitellogenesis in the Migratory Locust

    PubMed Central

    Song, Jiasheng; Jiang, Feng; Wang, Zhiming; Deng, Shun; Walker, Virginia K.; Zhou, Shutang

    2014-01-01

    Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation. PMID:25340846

  19. Juvenile hormone-receptor complex acts on mcm4 and mcm7 to promote polyploidy and vitellogenesis in the migratory locust.

    PubMed

    Guo, Wei; Wu, Zhongxia; Song, Jiasheng; Jiang, Feng; Wang, Zhiming; Deng, Shun; Walker, Virginia K; Zhou, Shutang

    2014-10-01

    Juvenile hormone (JH), a sesquiterpenoid produced by the corpora allata, coordinates insect growth, metamorphosis, and reproduction. While JH action for the repression of larval metamorphosis has been well studied, the molecular basis of JH in promoting adult reproduction has not been fully elucidated. Methoprene-tolerant (Met), the JH receptor, has been recently shown to mediate JH action during metamorphosis as well as in vitellogenesis, but again, the precise mechanism underlying the latter has been lacking. We have now demonstrated using Met RNAi to phenocopy a JH-deprived condition in migratory locusts, that JH stimulates DNA replication and increases ploidy in preparation for vitellogenesis. Mcm4 and Mcm7, two genes in the DNA replication pathway were expressed in the presence of JH and Met. Depletion of Mcm4 or Mcm7 inhibited de novo DNA synthesis and polyploidization, and resulted in the substantial reduction of vitellogenin mRNA levels as well as severely impaired oocyte maturation and ovarian growth. By using luciferase reporter and electrophoretic mobility shift assays, we have shown that Met directly regulates the transcription of Mcm4 and Mcm7 by binding to upstream consensus sequences with E-box or E-box-like motifs. Our work suggests that the JH-receptor complex acts on Mcm4 and Mcm7 to regulate DNA replication and polyploidy for vitellogenesis and oocyte maturation. PMID:25340846

  20. Identification of a monoclonal antibody against the leptin receptor that acts as an antagonist and blocks human monocyte and T cell activation.

    PubMed

    Fazeli, Mehdi; Zarkesh-Esfahani, Hamid; Wu, Zida; Maamra, Mabrouka; Bidlingmaier, Martin; Pockley, A Graham; Watson, Philip; Matarese, Giuseppe; Strasburger, Christian J; Ross, Richard J M

    2006-05-30

    Nutritional status has a major impact on the immune response and this is in part mediated by leptin, a pro-inflammatory cytokine. Preliminary data suggest that antagonism of leptin may offer a therapeutic approach for the treatment of some inflammatory disorders. We have tested monoclonal antibodies (mAbs) to the human leptin receptor (ObR) for antagonist activity using a leptin signalling bioassay. We identified a mAb, 9F8, which demonstrated dose-dependent antagonist activity in the leptin bioassay. Specificity of the mAb for ObR was confirmed using a plate binding assay. The 9F8 mAb displaced leptin binding to human ObR and enzymatically generated Fab fragments of 9F8 retained antagonist activity. Therefore the Fab fragment of 9F8 was cloned and recombinant 9F8 Fab (rFab) was purified from E. coli periplasmic fraction using a C-terminal His tag. Purified 9F8 rFab bound to human ObR and exhibited leptin antagonist activity. In vitro studies demonstrated that the 9F8 mAb inhibited leptin induced TNF-alpha production from human monocytes and anti-CD3 mAb induced proliferation of human T cells in PBMC culture. In conclusion, this study has identified a mAb to the human leptin receptor which inhibits leptin signalling and acts as a leptin antagonist in vitro. PMID:16690078

  1. ( sup 3 H)(D-PEN sup 2 , D-PEN sup 5 ) enkephalin binding to delta opioid receptors on intact neuroblastoma-glioma (NG 108-15) hybrid cells

    SciTech Connect

    Knapp, R.J.; Yamamura, H.I. )

    1990-01-01

    ({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin binding to intact NG 108-15 cells has been measured under physiological conditions of temperature and medium. The dissociation constant, receptor density, and Hill slope values measured under these conditions are consistent with values obtained by others using membranes prepared from these cells. Kinetic analysis of the radioligand binding to these cells show biphasic association and monophasic dissociation processes suggesting the presence of different receptor affinity states for the agonist. The data show that the binding affinity of ({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin under physiological conditions is not substantially different to that measured in 50 mM Tris buffer using cell membrane fractions. Unlike DPDPE, the {mu} opioid agonists morphine, normorphine, PL-17, and DAMGO, have much lower affinity for the {delta} receptor measured under these conditions than is observed by studies using 50 mM Tris buffer. The results described here suggest that this assay may serve as a useful model of {delta} opioid receptor binding in vivo.

  2. Roles of different subtypes of opioid receptors in mediating the ventrolateral orbital cortex opioid-induced inhibition of mirror-neuropathic pain in the rat.

    PubMed

    Zhao, M; Wang, J Y; Jia, H; Tang, J S

    2007-02-23

    Previous studies have demonstrated that opioid receptors in the prefrontal ventrolateral orbital cortex (VLO) are involved in anti-nociception. The aim of this current study was to examine whether opioid receptors in the VLO have effects on the hypersensitivity induced by contralateral L5 and L6 spinal nerve ligation (SNL), termed as mirror neuropathic pain (MNP) in the male rat. Morphine (1.0, 2.5, 5.0 microg) microinjected into the VLO contralateral to the SNL depressed the mechanical paw withdrawal assessed by von Frey filaments and the cold plate (4 degrees C)-induced paw lifting in a dose-dependent manner on the side without SNL. These effects were antagonized by microinjection of the non-selective opioid receptor antagonist naloxone (1.0 mug) into the same VLO site. Microinjection of endomorphin-1 (5.0 microg), a highly selective mu-opioid receptor agonist, and [d-Ala(2), d-Leu(5)]-enkephalin (DADLE, 10 microg), a delta-/mu-receptor agonist, also depressed the MNP. The effects of both drugs were blocked by selective mu-receptor antagonist beta-funaltrexamine (beta-FNA, 3.75 microg), but the effect of the DADLE was not influenced by the selective delta-receptor antagonist naltrindole (5.0 microg). Microinjection of the kappa-opioid receptor agonist spiradoline mesylate salt (U-62066) (100 microg) had no effect on the MNP. These results suggest that the VLO is involved in opioid-induced inhibition of the MNP and the effect is mediated by mu- (but not delta- and kappa-) opioid receptors. PMID:17184926

  3. Differential antagonism of tetramethylenedisulfotetramine-induced seizures by agents acting at NMDA and GABA{sub A} receptors

    SciTech Connect

    Shakarjian, Michael P.; Velíšková, Jana; Stanton, Patric K.; Velíšek, Libor

    2012-11-15

    Tetramethylenedisulfotetramine (TMDT) is a highly lethal neuroactive rodenticide responsible for many accidental and intentional poisonings in mainland China. Ease of synthesis, water solubility, potency, and difficulty to treat make TMDT a potential weapon for terrorist activity. We characterized TMDT-induced convulsions and mortality in male C57BL/6 mice. TMDT (ip) produced a continuum of twitches, clonic, and tonic–clonic seizures decreasing in onset latency and increasing in severity with increasing dose; 0.4 mg/kg was 100% lethal. The NMDA antagonist, ketamine (35 mg/kg) injected ip immediately after the first TMDT-induced seizure, did not change number of tonic–clonic seizures or lethality, but increased the number of clonic seizures. Doubling the ketamine dose decreased tonic–clonic seizures and eliminated lethality through a 60 min observation period. Treating mice with another NMDA antagonist, MK-801, 0.5 or 1 mg/kg ip, showed similar effects as low and high doses of ketamine, respectively, and prevented lethality, converting status epilepticus EEG activity to isolated interictal discharges. Treatment with these agents 15 min prior to TMDT administration did not increase their effectiveness. Post-treatment with the GABA{sub A} receptor allosteric enhancer diazepam (5 mg/kg) greatly reduced seizure manifestations and prevented lethality 60 min post-TMDT, but ictal events were evident in EEG recordings and, hours post-treatment, mice experienced status epilepticus and died. Thus, TMDT is a highly potent and lethal convulsant for which single-dose benzodiazepine treatment is inadequate in managing electrographic seizures or lethality. Repeated benzodiazepine dosing or combined application of benzodiazepines and NMDA receptor antagonists is more likely to be effective in treating TMDT poisoning. -- Highlights: ► TMDT produces convulsions and lethality at low doses in mice. ► Diazepam pre- or post-treatments inhibit TMDT-induced convulsions and death

  4. The brominated flame retardants TBP-AE and TBP-DBPE antagonize the chicken androgen receptor and act as potential endocrine disrupters in chicken LMH cells.

    PubMed

    Asnake, Solomon; Pradhan, Ajay; Kharlyngdoh, Joubert Banjop; Modig, Carina; Olsson, Per-Erik

    2015-12-01

    Increased exposure of birds to endocrine disrupting compounds has resulted in developmental and reproductive dysfunctions. We have recently identified the flame retardants, allyl-2,4,6-tribromophenyl ether (TBP-AE), 2-3-dibromopropyl-2,4,6-tribromophenyl ether (TBP-DBPE) and the TBP-DBPE metabolite 2-bromoallyl-2,4,6-tribromophenyl ether (TBP-BAE) as antagonists to both the human androgen receptor (AR) and the zebrafish AR. In the present study, we aimed at determining whether these compounds also interact with the chicken AR. In silico modeling studies showed that TBP-AE, TBP-BAE and TBP-DBPE were able to dock into to the chicken AR ligand-binding pocket. In vitro transfection assays revealed that all three brominated compounds acted as chicken AR antagonists, inhibiting testosterone induced AR activation. In addition, qRT-PCR studies confirmed that they act as AR antagonists and demonstrated that they also alter gene expression patterns of apoptotic, anti-apoptotic, drug metabolizing and amino acid transporter genes. These studies, using chicken LMH cells, suggest that TBP-AE, TBP-BAE and TBP-DBPE are potential endocrine disrupters in chicken. PMID:26318274

  5. Clobetasol and Halcinonide Act as Smoothened Agonists to Promote Myelin Gene Expression and RxRγ Receptor Activation

    PubMed Central

    De Nardis, Velia; Di Giandomenico, Daniele; Lucisano, Giuseppe; Scardapane, Marco; Poma, Anna; Ragnini-Wilson, Antonella

    2015-01-01

    One of the causes of permanent disability in chronic multiple sclerosis patients is the inability of oligodendrocyte progenitor cells (OPCs) to terminate their maturation program at lesions. To identify key regulators of myelin gene expression acting at the last stages of OPC maturation we developed a drug repositioning strategy based on the mouse immortalized oligodendrocyte (OL) cell line Oli-neu brought to the premyelination stage by stably expressing a key factor regulating the last stages of OL maturation. The Prestwick Chemical Library® of 1,200 FDA-approved compound(s) was repositioned at three dosages based on the induction of Myelin Basic Protein (MBP) expression. Drug hits were further validated using dosage-dependent reproducibility tests and biochemical assays. The glucocorticoid class of compounds was the most highly represented and we found that they can be divided in three groups according to their efficacy on MBP up-regulation. Since target identification is crucial before bringing compounds to the clinic, we searched for common targets of the primary screen hits based on their known chemical-target interactomes, and the pathways predicted by top ranking compounds were validated using specific inhibitors. Two of the top ranking compounds, Halcinonide and Clobetasol, act as Smoothened (Smo) agonists to up-regulate myelin gene expression in the Oli-neuM cell line. Further, RxRγ activation is required for MBP expression upon Halcinonide and Clobetasol treatment. These data indicate Clobetasol and Halcinonide as potential promyelinating drugs and also provide a mechanistic understanding of their mode of action in the pathway leading to myelination in OPCs. Furthermore, our classification of glucocorticoids with respect to MBP expression provides important novel insights into their effects in the CNS and a rational criteria for their choice in combinatorial therapies in de-myelinating diseases. PMID:26658258

  6. RABA Members Act in Distinct Steps of Subcellular Trafficking of the FLAGELLIN SENSING2 Receptor[W

    PubMed Central

    Choi, Seung-won; Tamaki, Takayuki; Ebine, Kazuo; Uemura, Tomohiro; Ueda, Takashi; Nakano, Akihiko

    2013-01-01

    Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo–synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endo- and exocytosis. PMID:23532067

  7. 4,5-Substituted 3-Isoxazolols with Insecticidal Activity Act as Competitive Antagonists of Housefly GABA Receptors.

    PubMed

    Liu, Genyan; Ozoe, Fumiyo; Furuta, Kenjiro; Ozoe, Yoshihisa

    2015-07-22

    The insect GABA receptor (GABAR), which is composed of five RDL subunits, represents an important target for insecticides. A series of 4,5-disubstituted 3-isoxazolols, including muscimol analogues, were synthesized and examined for their activities against four splice variants (ac, ad, bc, and bd) of housefly GABARs expressed in Xenopus oocytes. Muscimol was a more potent agonist than GABA in all four splice variants, whereas synthesized analogues did not exhibit agonism but rather antagonism in housefly GABARs. The introduction of bicyclic aromatic groups at the 4-position of muscimol and the simultaneous replacement of the aminomethyl group with a carbamoyl group at the 5-position to afford six 4-aryl-5-carbamoyl-3-isoxazolols resulted in compounds that exhibited significantly enhanced antagonism with IC50 values in the low micromolar range in the ac variant. The inhibition of GABA-induced currents by 100 μM analogues was approximately 1.5-4-fold greater in the ac and bc variants than in the ad and bd variants. 4-(3-Biphenylyl)-5-carbamoyl-3-isoxazolol displayed competitive antagonism, with IC50 values of 30, 34, 107, and 96 μM in the ac, bc, ad, and bd variants, respectively, and exhibited moderate insecticidal activity against houseflies, with an LD50 value of 5.6 nmol/fly. These findings suggest that these 3-isoxazolol analogues are novel lead compounds for the design and development of insecticides that target the orthosteric site of housefly GABARs. PMID:26120732

  8. Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

    PubMed

    Peng, Jian; Tao, Xin; Li, Rui; Hu, Jingru; Ruan, Jie; Wang, Ruihua; Yang, Manyi; Yang, Rirong; Dong, Xiangru; Chen, Shangwu; Xu, Anlong; Yuan, Shaochun

    2015-10-01

    Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate. PMID:26324776

  9. Synthetic retinoid CD437 induces apoptosis and acts synergistically with TRAIL receptor-2 agonist in malignant melanoma.

    PubMed

    Magnussen, Gry Irene; Ree Rosnes, Anne Katrine; Shahzidi, Susan; Dong, Hiep Phuc; Emilsen, Elisabeth; Engesæter, Birgit; Flørenes, Vivi Ann

    2012-04-13

    The novel synthetic retinoid, CD437, shows potent anti-tumor activity in a range of different cancer cell lines and now serves as a prototype for development of new retinoid related molecules (RRMs). The purpose of this study was to examine the effect and cellular targets of CD437 in the human metastatic melanoma cell lines FEMX-1 and WM239. We showed that treatment with CD437 led to cell cycle arrest and induced apoptosis through both the extrinsic- and intrinsic pathways (caspase 8, -9 and PARP cleavage) in both cell lines. Interestingly, apoptosis was induced independently of DNA-fragmentation in FEMX-1 cells, and appeared partially caspase-independent in the WM239 cells. Additionally, up-regulation of CHOP mRNA and cathepsin D protein expression, following retinoid treatment, suggests involvement of the endoplasmatic reticulum (ER) and lysosomes, respectively. Combination of suboptimal concentrations of CD437 and lexatumumab, a TRAIL death receptor-2 agonist, resulted in synergistic reduction of viable cells, along with increased PARP cleavage. These results indicate that CD437 has a strong anti-neoplastic effect alone and in combination with lexatumumab in melanoma cell lines. PMID:22446330

  10. A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus

    PubMed Central

    Shimizu, Nobuaki; Tanaka, Atsushi; Mori, Takahisa; Ohtsuki, Takahiro; Hoque, Aliful; Jinno-Oue, Atsushi; Apichartpiyakul, Chatchawann; Kusagawa, Shigeru; Takebe, Yutaka; Hoshino, Hiroo

    2008-01-01

    Background More than 10 members of seven-transmembrane G protein-coupled receptors (GPCRs) have been shown to work as coreceptors for human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and simian immunodeficiency viruses (SIVs). As a common feature of HIV/SIV coreceptors, tyrosine residues are present with asparagines, aspartic acids or glutamic acids in the amino-terminal extracellular regions (NTRs). We noticed that a receptor for N-formylpeptides, FPRL1, also contains two tyrosine residues accompanied by glutamic acids in its NTR. It was reported that monocytes expressing CCR5 and FPRL1 in addition to CD4 are activated by treatment with ligands or agonists of FPRL1. Activated monocytes down-modulate CCR5 and become resistant to infection by HIV-1 strains. Thus, FPRL1 plays important roles in protection of monocyptes against HIV-1 infection. However, its own coreceptor activity has not been elucidated yet. In this study, we examined coreceptor activities of FPRL1 for HIV/SIV strains including primary HIV-1 isolates. Results A CD4-transduced human glioma cell line, NP-2/CD4, is strictly resistant to HIV/SIV infection. We have reported that when NP-2/CD4 cells are transduced with a GPCR having coreceptor activity, the cells become susceptible to HIV/SIV strains. When NP-2/CD4 cells were transduced with FPRL1, the resultant NP-2/CD4/FPRL1 cells became markedly susceptible to some laboratory-adapted HIV/SIV strains. We found that FPRL1 is also efficiently used as a coreceptor by primary HIV-1 isolates as well as CCR5 or CXCR4. Amino acid sequences linked to the FPRL1 use could not be detected in the V3 loop of the HIV-1 Env protein. Coreceptor activities of FPRL1 were partially blocked by the forymyl-Met-Leu-Phe (fMLF) peptide. Conclusion We conclude that FPRL1 is a novel and efficient coreceptor for HIV/SIV strains. FPRL1 works as a bifunctional factor in HIV-1 infection. Namely, the role of FPRL1 in HIV-1 infection is protective and/or promotive in

  11. Calcium sensing receptor function supports osteoblast survival and acts as a co-factor in PTH anabolic actions in bone

    PubMed Central

    Al-Dujaili, Saja A.; Koh, Amy J.; Dang, Ming; Mi, Xue; Chang, Wenhan; Ma, Peter X.; McCauley, Laurie K.

    2016-01-01

    Anabolic actions of PTH in bone involve increased deposition of mineralizing matrix. Regulatory feedback of the process may be important to maintain calcium homeostasis and, in turn, calcium may inform the process. This investigation clarified the role of calcium availability and the calcium sensing receptor (CaSR) in the anabolic actions of PTH. CaSR function promoted osteoblastic cell numbers, with lower cell numbers in post-confluent cultures of primary calvarial cells from Col1-CaSR knock-out (KO) mice, and for calvarial cells from wild-type (WT) mice treated with a calcilytic. Increased apoptosis of calvarial cells with calcilytic treatment suggested CaSR is critical for protection against stage-dependent cell death. Whole and cortical, but not trabecular, bone parameters were significantly lower in Col1-CaSR KO mice versus WT littermates. Intact Col1-CaSR KO mice had lower serum P1NP levels relative to WT. PTH treatment displayed anabolic actions in WT and, to a lesser degree, KO mice, and rescued the lower P1NP levels in KO mice. Furthermore, PTH effects on whole tibiae were inhibited by osteoblast-specific CaSR ablation. Vertebral body implants (vossicles) from untreated Col1-CaSR KO and WT mice had similar bone volumes after 4 weeks of implantation in athymic mice. These findings suggest that trabecular bone formation can occur independently of the CaSR, and that the CaSR plays a collaborative role in the PTH anabolic effects on bone. PMID:26579618

  12. Calcium Sensing Receptor Function Supports Osteoblast Survival and Acts as a Co-Factor in PTH Anabolic Actions in Bone.

    PubMed

    Al-Dujaili, Saja A; Koh, Amy J; Dang, Ming; Mi, Xue; Chang, Wenhan; Ma, Peter X; McCauley, Laurie K

    2016-07-01

    Anabolic actions of PTH in bone involve increased deposition of mineralizing matrix. Regulatory feedback of the process may be important to maintain calcium homeostasis and, in turn, calcium may inform the process. This investigation clarified the role of calcium availability and the calcium sensing receptor (CaSR) in the anabolic actions of PTH. CaSR function promoted osteoblastic cell numbers, with lower cell numbers in post-confluent cultures of primary calvarial cells from Col1-CaSR knock-out (KO) mice, and for calvarial cells from wild-type (WT) mice treated with a calcilytic. Increased apoptosis of calvarial cells with calcilytic treatment suggested CaSR is critical for protection against stage-dependent cell death. Whole and cortical, but not trabecular, bone parameters were significantly lower in Col1-CaSR KO mice versus WT littermates. Intact Col1-CaSR KO mice had lower serum P1NP levels relative to WT. PTH treatment displayed anabolic actions in WT and, to a lesser degree, KO mice, and rescued the lower P1NP levels in KO mice. Furthermore, PTH effects on whole tibiae were inhibited by osteoblast-specific CaSR ablation. Vertebral body implants (vossicles) from untreated Col1-CaSR KO and WT mice had similar bone volumes after 4 weeks of implantation in athymic mice. These findings suggest that trabecular bone formation can occur independently of the CaSR, and that the CaSR plays a collaborative role in the PTH anabolic effects on bone. J. Cell. Biochem. 117: 1556-1567, 2016. © 2015 Wiley Periodicals, Inc. PMID:26579618

  13. Identification of opioid receptor subtypes in antinociceptive actions of supraspinally-administered mitragynine in mice.

    PubMed

    Thongpradichote, S; Matsumoto, K; Tohda, M; Takayama, H; Aimi, N; Sakai, S; Watanabe, H

    1998-01-01

    Mitragynine (MG), a major alkaloidal constituent extracted from the plant Mitragyna speciosa Korth, is known to exert an opioid-like activity. Our previous study showed the involvement of opioid systems in the antinociceptive activity of MG in the tail-pinch and hot-plate tests in mice. In the present study, to clarify the opioid receptor subtypes involved in the antinociceptive action of MG, we investigated the effects of selective antagonists for mu-, delta- and kappa- opioid receptors on antinociception caused by the intracerebroventricular (i.c.v.) injection of MG in the tail-pinch and hot-plate tests in mice. The coadministration of a selective mu-opioid antagonist, cyprodime (1-10 microg, i.c.v.) and the pretreatment with a selective mu1-opioid antagonist naloxonazine (1-3 microg, i.c.v.) significantly antagonized the antinociceptive activities of MG (10 microg, i.c.v.) and morphine (MOR, 3 microg, i.c.v.) in the tail-pinch and hot-plate tests. Naltrindole (1-5 ng, i.c.v.), a selective delta-opioid antagonist, also blocked the effects of MG (10 microg, i.c.v.) without affecting MOR (3 microg, i.c.v.) antinociception. Nor-binaltorphimine, a selective kappa-opioid antagonist, significantly attenuated MG (10 microg, i.c.v.) antinociception in the tail-pinch test but not in the hot-plate test at the dose (1 microg, i.c.v.) that antagonized the antinociceptive effects of the selective kappa-opioid agonist U50,488H in both tests, while it had no effect on MOR antinociception in either tests. These results suggest that antinociception caused by i.c.v. MG is dominantly mediated by mu- and delta-opioid receptor subtypes, and that the selectivity of MG for the supraspinal opioid receptor subtypes differs from that of MOR in mice. PMID:9585164

  14. The σ1 Receptor Engages the Redox-Regulated HINT1 Protein to Bring Opioid Analgesia Under NMDA Receptor Negative Control

    PubMed Central

    Rodríguez-Muñoz, María; Sánchez-Blázquez, Pilar; Herrero-Labrador, Raquel; Martínez-Murillo, Ricardo; Merlos, Manuel; Vela, José Miguel

    2015-01-01

    Abstract Aims: The in vivo pharmacology of the sigma 1 receptor (σ1R) is certainly complex; however, σ1R antagonists are of therapeutic interest, because they enhance mu-opioid receptor (MOR)-mediated antinociception and reduce neuropathic pain. Thus, we investigated whether the σ1R is involved in the negative control that glutamate N-methyl-d-aspartate acid receptors (NMDARs) exert on opioid antinociception. Results: The MOR C terminus carries the histidine triad nucleotide-binding protein 1 (HINT1) coupled to the regulator of G-protein signaling RGSZ2-neural nitric oxide synthase assembly. Activated MORs stimulate the production of nitric oxide (NO), and the redox zinc switch RGSZ2 converts this signal into free zinc ions that are required to recruit the redox sensor PKCγ to HINT1 proteins. Then, PKCγ impairs HINT1-RGSZ2 association and enables σ1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. The inhibition of NOS or the absence of σ1Rs prevents HINT1-PKCγ interaction, and MOR-NMDAR cross-regulation fails. The σ1R antagonists transitorily remove the binding of σ1Rs to NR1 subunits, facilitate the entrance of negative regulators of NMDARs, likely Ca2+-CaM, and prevent NR1 interaction with HINT1, thereby impairing the negative feedback of glutamate on opioid analgesia. Innovation: A redox-regulated process situates MOR signaling under NMDAR control, and in this context, the σ1R binds to the cytosolic C terminal region of the NMDAR NR1 subunit. Conclusion: The σ1R antagonists enhance opioid analgesia in naïve mice by releasing MORs from the negative influence of NMDARs, and they also reset antinociception in morphine tolerant animals. Moreover, σ1R antagonists alleviate neuropathic pain, probably by driving the inhibition of up-regulated NMDARs. Antioxid. Redox Signal. 22, 799–818. PMID:25557043

  15. HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation

    PubMed Central

    Galvani, Sylvain; Sanson, Marie; Blaho, Victoria A.; Swendeman, Steven L.; Obinata, Hideru; Conger, Heather; Dahlbäck, Björn; Kono, Mari; Proia, Richard L.; Smith, Jonathan D.; Hla, Timothy

    2016-01-01

    The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S1P1 in adult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, was enhanced in mice with endothelial cell–specific deletion of S1pr1 and suppressed in mice with endothelial cell–specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. The chaperones ApoM+HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; therefore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM+HDL-S1P, but not to albumin-S1P, promoted the formation of a cell surface S1P1–β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-1 abundance. Although S1P bound to either chaperone induced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelial cell–specific deletion of S1pr1 in the hypercholesterolemic Apoe−/− mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM+HDL to act as a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL. PMID:26268607

  16. Bryostatin-1, a Naturally Occurring Antineoplastic Agent, Acts as a Toll-like Receptor 4 (TLR-4) Ligand and Induces Unique Cytokines and Chemokines in Dendritic Cells*

    PubMed Central

    Ariza, Maria Eugenia; Ramakrishnan, Rupal; Singh, Narendra P.; Chauhan, Ashok; Nagarkatti, Prakash S.; Nagarkatti, Mitzi

    2011-01-01

    Bryostatin-1 (Bryo-1), a natural macrocyclic lactone, is clinically used as an anti-cancer agent. In this study, we demonstrate for the first time that Bryo-1 acts as a Toll-like receptor 4 (TLR4) ligand. Interestingly, activation of bone marrow-derived dendritic cells (in vitro with Bryo-1) led to a TLR4-dependent biphasic activation of nuclear factor-κB (NF-κB) and the unique induction of cytokines (IL-5, IL-6, and IL-10) and chemokines, including RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein 1α (MIP1-α). In addition, EMSA demonstrated that Bryo-1-mediated induction of RANTES was regulated by NF-κB and the interferon regulatory factors (IRF)-1, IRF-3, and IRF-7 to the RANTES independently of myeloid differentiation primary response gene-88 (MyD88). Bryo-1 was able to induce the transcriptional activation of IRF-3 through the TLR4/MD2-dependent pathway. In vivo administration of Bryo-1 triggered a TLR-4-dependent T helper cell 2 (Th2) cytokine response and expanded a subset of myeloid dendritic cells that expressed a CD11chighCD8α− CD11b+CD4+ phenotype. This study demonstrates that Bryo-1 can act as a TLR4 ligand and activate innate immunity. Moreover, the ability of Bryo-1 to trigger RANTES and MIP1-α suggests that Bryo-1 could potentially be used to prevent HIV-1 infection. Finally, induction of a Th2 response by Bryo-1 may help treat inflammatory diseases mediated by Th1 cells. Together, our studies have a major impact on the clinical use of Bryo-1 as an anti-cancer and immunopotentiating agent. PMID:21036898

  17. Two Galpha(i1) rate-modifying mutations act in concert to allow receptor-independent, steady-state measurements of RGS protein activity.

    PubMed

    Zielinski, Thomas; Kimple, Adam J; Hutsell, Stephanie Q; Koeff, Mark D; Siderovski, David P; Lowery, Robert G

    2009-12-01

    RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate Galpha subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from Galpha and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a Galpha(i1) mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Galpha(i1)(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on Galpha(i) subunits and not affected by those unable to act on Galpha(i), demonstrating that the Galpha/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of Galpha( i1)(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on Galpha(i1)(R178M/A326S), the authors developed a Transcreener fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Galpha(i1)(R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling. PMID:19820068

  18. Dynorphin activation of kappa opioid receptor reduces neuronal excitability in the paraventricular nucleus of mouse thalamus.

    PubMed

    Chen, Zhiheng; Tang, Yamei; Tao, Huai; Li, Cunyan; Zhang, Xianghui; Liu, Yong

    2015-10-01

    It has been reported that kappa opioid receptor (KOR) is expressed in the paraventricular nucleus of thalamus (PVT), a brain region associated with arousal, drug reward and stress. Although intra-PVT infusion of KOR agonist was found to inhibit drug-seeking behavior, it is still unclear whether endogenous KOR agonists directly regulate PVT neuron activity. Here, we investigated the effect of the endogenous KOR agonist dynorphin-A (Dyn-A) on the excitability of mouse PVT neurons at different developmental ages. We found Dyn-A strongly inhibited PVT neurons through a direct postsynaptic hyperpolarization. Under voltage-clamp configuration, Dyn-A evoked an obvious outward current in majority of neurons tested in anterior PVT (aPVT) but only in minority of neurons in posterior PVT (pPVT). The Dyn-A current was abolished by KOR antagonist nor-BNI, Ba(2+) and non-hydrolyzable GDP analogue GDP-β-s, indicating that Dyn-A activates KOR and opens G-protein-coupled inwardly rectifying potassium channels in PVT neurons. More interestingly, by comparing Dyn-A currents in aPVT neurons of mice at various ages, we found Dyn-A evoked significant larger current in aPVT neurons from mice around prepuberty and early puberty stage. In addition, KOR activation by Dyn-A didn't produce obvious desensitization, while mu opioid receptor (MOR) activation induced obvious desensitization of mu receptor itself and also heterologous desensitization of KOR in PVT neurons. Together, our findings indicate that Dyn-A activates KOR and inhibits aPVT neurons in mice at various ages especially around puberty, suggesting a possible role of KOR in regulating aPVT-related brain function including stress response and drug-seeking behavior during adolescence. PMID:26056031

  19. G Protein Beta 5 Is Targeted to D2-Dopamine Receptor-Containing Biochemical Compartments and Blocks Dopamine-Dependent Receptor Internalization

    PubMed Central

    Octeau, J. Christopher; Schrader, Joseph M.; Masuho, Ikuo; Sharma, Meenakshi; Aiudi, Christopher; Chen, Ching-Kang; Kovoor, Abraham; Celver, Jeremy

    2014-01-01

    G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins. PMID:25162404

  20. G protein beta 5 is targeted to D2-dopamine receptor-containing biochemical compartments and blocks dopamine-dependent receptor internalization.

    PubMed

    Octeau, J Christopher; Schrader, Joseph M; Masuho, Ikuo; Sharma, Meenakshi; Aiudi, Christopher; Chen, Ching-Kang; Kovoor, Abraham; Celver, Jeremy

    2014-01-01

    G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins. PMID:25162404

  1. [Preparation and the biological effect of fusion protein GLP-1-exendin-4/ IgG4(Fc) fusion protein as long acting GLP-1 receptor agonist].

    PubMed

    Zheng, Yun-cheng

    2015-12-01

    GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for treatment of diabetes due to its short half-life (t½, 2-5 min). Exendin-4 is a polypeptide isolated from lizard saliva, which can bind to GLP-1 receptor, produce physiological effects similar to GLP-1, t½ up to 2.5 h, therefore, we developed a long-lasting GLP-1 receptor agonists and GLP-1-exendin-4 fusion IgG4 Fc [GLP-1-exendin-4/ IgG4(Fc)]. We constructed the eukaryotic expression vector of human GLP-1-exendin-4/IgG4(Fc)-pOptiVEC- TOPO by gene recombination technique and expressed the fusion protein human GLP-1-IgG4 (Fc) in CHO/DG44 cells. The fusion protein stimulated the INS-1 cells secretion of insulin, GLP-1, exendin-4 and fusion protein in CD1 mice pharmacokinetic experiments, as well as GLP-1, exendin-4 and fusion protein did anti-diabetic effect on streptozotocin induced mice. Results demonstrated that the GLP-1-exendin-4/IgG4(Fc) positive CHO/DG44 clones were chosen and the media from these positive clones. Western blotting showed that one protein band was found to match well with the predicted relative molecular mass of human GLP-1-exendin-4/IgG4(Fc). Insulin RIA showed that GLP-1-exendin-4/IgG4(Fc) dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (ip), the fusion protein peaked at 30 min in circulation and maintained a plateau for 200 h. Natural biological half-life of exendin-4 was (1.39 ± 0.28) h, GLP-1 in vivo t½ 4 min, indicating that fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1-exendin-4/IgG4(Fc) was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced diabetes in mice, longer duration of the biological activity of the fusion protein. The biological activity was significantly higher than that of GLP-1 and exendin-4. GLP-1-exendin-4/IgG4(Fc) has good anti-diabetic activity

  2. Human SLURP-1 and SLURP-2 Proteins Acting on Nicotinic Acetylcholine Receptors Reduce Proliferation of Human Colorectal Adenocarcinoma HT-29 Cells.

    PubMed

    Lyukmanova, E N; Shulepko, M A; Bychkov, M L; Shenkarev, Z O; Paramonov, A S; Chugunov, A O; Arseniev, A S; Dolgikh, D A; Kirpichnikov, M P

    2014-10-01

    Human secreted Ly-6/uPAR related proteins (SLURP-1 and SLURP-2) are produced by various cells, including the epithelium and immune system. These proteins act as autocrine/paracrine hormones regulating the growth and differentiation of keratinocytes and are also involved in the control of inflammation and malignant cell transformation. These effects are assumed to be mediated by the interactions of SLURP-1 and SLURP-2 with the α7 and α3β2 subtypes of nicotinic acetylcholine receptors (nAChRs), respectively. Available knowledge about the molecular mechanism underling the SLURP-1 and SLURP-2 effects is very limited. SLURP-2 remains one of the most poorly studied proteins of the Ly-6/uPAR family. In this study, we designed for the first time a bacterial system for SLURP-2 expression and a protocol for refolding of the protein from cytoplasmic inclusion bodies. Milligram quantities of recombinant SLURP-2 and its 13C-15N-labeled analog were obtained. The recombinant protein was characterized by NMR spectroscopy, and a structural model was developed. A comparative study of the SLURP-1 and SLURP-2 effects on the epithelial cell growth was conducted using human colorectal adenocarcinoma HT-29 cells, which express only α7-nAChRs. A pronounced antiproliferative effect of both proteins was observed. Incubation of cells with 1 μM SLURP-1 and 1 μM SLURP-2 during 48 h led to a reduction in the cell number down to ~ 54 and 63% relative to the control, respectively. Fluorescent microscopy did not reveal either apoptotic or necrotic cell death. An analysis of the dose-response curve revealed the concentration-dependent mode of the SLURP-1 and SLURP-2 action with EC50 ~ 0.1 and 0.2 nM, respectively. These findings suggest that the α7-nAChR is the main receptor responsible for the antiproliferative effect of SLURP proteins in epithelial cells. PMID:25558396

  3. Stimulation of serotonin2C receptors elicits abnormal oral movements by acting on pathways other than the sensorimotor one in the rat basal ganglia.

    PubMed

    Beyeler, A; Kadiri, N; Navailles, S; Boujema, M Ben; Gonon, F; Moine, C Le; Gross, C; De Deurwaerdère, P

    2010-08-11

    Serotonin2C (5-HT(2C)) receptors act in the basal ganglia, a group of sub-cortical structures involved in motor behavior, where they are thought to modulate oral activity and participate in iatrogenic motor side-effects in Parkinson's disease and Schizophrenia. Whether abnormal movements initiated by 5-HT(2C) receptors are directly consequent to dysfunctions of the motor circuit is uncertain. In the present study, we combined behavioral, immunohistochemical and extracellular single-cell recordings approaches in rats to investigate the effect of the 5-HT(2C) agonist Ro-60-0175 respectively on orofacial dyskinesia, the expression of the marker of neuronal activity c-Fos in basal ganglia and the electrophysiological activity of substantia nigra pars reticulata (SNr) neuron connected to the orofacial motor cortex (OfMC) or the medial prefrontal cortex (mPFC). The results show that Ro-60-0175 (1 mg/kg) caused bouts of orofacial movements that were suppressed by the 5-HT(2C) antagonist SB-243213 (1 mg/kg). Ro-60-0175 (0.3, 1, 3 mg/kg) dose-dependently enhanced Fos expression in the striatum and the nucleus accumbens. At the highest dose, it enhanced Fos expression in the subthalamic nucleus, the SNr and the entopeduncular nucleus but not in the external globus pallidus. However, the effect of Ro-60-0175 was mainly associated with associative/limbic regions of basal ganglia whereas subregions of basal ganglia corresponding to sensorimotor territories were devoid of Fos labeling. Ro-60-0175 (1-3 mg/kg) did not affect the electrophysiological activity of SNr neurons connected to the OfMC nor their excitatory-inhibitory-excitatory responses to the OfMC electrical stimulation. Conversely, Ro-60-0175 (1 mg/kg) enhanced the late excitatory response of SNr neurons evoked by the mPFC electrical stimulation. These results suggest that oral dyskinesia induced by 5-HT(2C) agonists are not restricted to aberrant signalling in the orofacial motor circuit and demonstrate discrete

  4. Morphine drives internal ribosome entry site-mediated hnRNP K translation in neurons through opioid receptor-dependent signaling

    PubMed Central

    Lee, Pin-Tse; Chao, Po-Kuan; Ou, Li-Chin; Chuang, Jian-Ying; Lin, Yen-Chang; Chen, Shu-Chun; Chang, Hsiao-Fu; Law, Ping-Yee; Loh, Horace H.; Chao, Yu-Sheng; Su, Tsung-Ping; Yeh, Shiu-Hwa

    2014-01-01

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the promoter region of mu-opioid receptor (MOR) to regulate its transcriptional activity. How hnRNP K contributes to the analgesic effects of morphine, however, is largely unknown. We provide evidence that morphine increases hnRNP K protein expression via MOR activation in rat primary cortical neurons and HEK-293 cells expressing MORs, without increasing mRNA levels. Using the bicistronic reporter assay, we examined whether morphine-mediated accumulation of hnRNP K resulted from translational control. We identified potential internal ribosome entry site elements located in the 5′ untranslated regions of hnRNP K transcripts that were regulated by morphine. This finding suggests that internal translation contributes to the morphine-induced accumulation of hnRNP K protein in regions of the central nervous system correlated with nociceptive and antinociceptive modulatory systems in mice. Finally, we found that down-regulation of hnRNP K mediated by siRNA attenuated morphine-induced hyperpolarization of membrane potential in AtT20 cells. Silencing hnRNP K expression in the spinal cord increased nociceptive sensitivity in wild-type mice, but not in MOR-knockout mice. Thus, our findings identify the role of translational control of hnRNP K in morphine-induced analgesia through activation of MOR. PMID:25361975

  5. Somatostatin receptor subtypes 2 and 5 are associated with better survival in operable hepatitis B-related hepatocellular carcinoma following octreotide long-acting release treatment

    PubMed Central

    LIU, YAO; JIANG, LI; MU, YI

    2013-01-01

    Liver resections for hepatocellular carcinoma (HCC) in cirrhotic livers are associated with early recurrence and poor survival. Somatostatin analogues (SSAs) have been reported to inhibit cell proliferation by interacting with specific somatostatin receptors (SSTRs) 2 and 5. The present study investigated whether SSTR expression in HCC was associated with the clinical outcome following octreotide long-acting release (LAR) treatment. Paired tumor and cirrhotic liver samples were obtained following a liver resection from 99 patients with stage I–II HCC and HBV-related cirrhosis. The expression of SSTR2 and 5 was assessed using quantitative (q)PCR and immunohistochemistry. The patients were classified into two groups, the high expression (n=47) and low expression (n=52) groups, based on the gene expression levels. The clinicopathological data and survival results of the two groups were compared. When compared with the surrounding cirrhotic tissue, the SSTR2 and 5 mRNA levels were significantly decreased in the HCC tissue. There were no significant differences between the groups with respect to the baseline characteristics. The tumor recurrence rate was significantly lower in the high expression group compared with that of the low expression group (63.83% vs. 82.69%; P=0.033). The 1-, 3- and 5-year disease-free and overall survival rates of the high expression group were 97, 89 and 71% and 98, 89 and 74%, respectively. The survival time of the members of the high expression group was longer compared with that of the low expression group. The multivariate analysis revealed that the TNM-7 stage and SSTR2 expression were independent prognostic factors for survival. In conclusion, SSTR mRNA expression correlated with survival in patients with early-stage hepatitis B virus (HBV)-related HCC who were treated with octreotide LAR following surgery. The inhibitory effects of SSAs on tumor growth may be mediated by SSTR expression. PMID:24137418

  6. The long-acting β2-adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M A

    2015-01-01

    Background and Purpose Inhaled glucocorticoid (ICS)/long-acting β2-adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. Experimental Approach BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. Key Results Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. Conclusions and Implications These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues. PMID:25598440

  7. Effects of orally-bioavailable short-acting kappa opioid receptor-selective antagonist LY2456302 on nicotine withdrawal in mice.

    PubMed

    Jackson, Kia J; Jackson, Asti; Carroll, F Ivy; Damaj, M Imad

    2015-10-01

    Kappa opioid receptor (KOR) signaling has been implicated in mediating behavioral and biochemical effects associated with drug dependence. The most commonly used KOR antagonists, norbinaltorphimine (norBNI) and (3R)-7-Hydroxy-N{(1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl}-1,2,3,4-tetrahydro-3-isoquinoline-carboxamide (JDTic), have provided a wealth of information in this area; however, the delayed onset and long-lasting effects of these antagonists complicate experimental design and interpretation of results, and make them less than ideal for clinical studies. Initial studies with the recently developed KOR antagonist, LY2456302, show that the compound is a short acting, high-affinity, selective KOR antagonist with therapeutic potential for mood disorders and ethanol use in animal models, and is well tolerated in humans. The goal of the current study was to evaluate the effectiveness of LY2456302 in alleviating the nicotine withdrawal syndrome in mice. Mice were chronically treated with nicotine for 14 days and physical and affective nicotine withdrawal signs were measured using a spontaneous nicotine withdrawal model and conditioned place aversion (CPA) following pre-treatment with LY2456302, administered orally. Vehicle treated nicotine withdrawn mice displayed significant anxiety-related behavior, somatic signs, hyperalgesia, and CPA. Similar to previous studies with norBNI and JDTic, LY2456302 alleviated the nicotine withdrawal syndrome, as evidenced by decreased expression of nicotine withdrawal induced anxiety-related behavior, somatic signs, and CPA, and increased hotplate latency in nicotine withdrawn mice following pre-treatment. Given the current results, and with its favorable pharmacokinetic and pharmacodynamic profile, LY2456302 may be a useful therapeutic agent for treatment of multiple aspects of the nicotine withdrawal syndrome. PMID:26044637

  8. Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Agonist, GLP-1-IgG2σ-Fc

    PubMed Central

    Yang, Yi; Chen, Fang; Wan, Deyou; Liu, Yunhui; Yang, Li; Feng, Hongru; Cui, Xinling; Gao, Xin; Song, Haifeng

    2016-01-01

    Human GLP-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes. However, its clinical benefits are limited by its short half-life, which is less than 2 min because of its small size and rapid enzymatic inactivation by dipeptidyl peptidase IV. We engineered GLP-1-IgG2σ-Fc, a 68-kDa fusion protein linking a variant human GLP-1 (A8G/G26E/R36G) to a human IgG2σ constant heavy-chain. A stably transfected Chinese hamster ovary cell line was obtained using electroporation. Western blotting showed that the expressed protein was immunoreactive to both GLP-1 and IgG antibodies. GLP-1-IgG2σ-Fc stimulated insulin secretion from INS-1 cells in a dose- and glucose-dependent manner and increased insulin mRNA expression. The half-life of GLP-1-IgG2σ-Fc in cynomolgus monkeys was approximately 57.1 ± 4.5 h. In the KKAy mouse model of diabetes, one intraperitoneal injection of GLP-1-IgG2σ-Fc (1 mg/kg) reduced blood glucose levels for 5 days. A 4-week repeat-administration study identified sustained effects on blood glucose levels. Oral glucose tolerance tests conducted at the beginning and end of this 4-week period showed that GLP-1-IgG2σ-Fc produced a stable glucose lowering effect. In addition, KKAy mice treated with GLP-1-IgG2σ-Fc showed statistically significant weight loss from day 23. In conclusion, these properties of GLP-1-IgG2σ-Fc demonstrated that it represented a potential long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes. PMID:27232339

  9. Modulation of spontaneous intracellular Ca²⁺ fluctuations and spontaneous cholinergic transmission in rat chromaffin cells in situ by endogenous GABA acting on GABAA receptors.

    PubMed

    Tzitzitlini, Alejandre-García; Pedro, Segura-Chama; Martha, Pérez-Armendáriz E; Rodolfo, Delgado-Lezama; Arturo, Hernández-Cruz

    2016-02-01

    Using fluorescence [Ca(2+)]i imaging in rat adrenal slices, we characterized the effects of agonists and antagonists of the GABAA receptor (GABAA-R) on resting intracellular Ca(2+) ([Ca(2+)]i) and spontaneous [Ca(2+)]i fluctuations (SCFs) in hundreds of individual chromaffin cells (CCs) recorded simultaneously in situ. Muscimol, a GABAA-R agonist (20 μM; 25 s), induced an increase of resting [Ca(2+)]i in 43 ± 3 % of CCs, a decrease in 26 ± 2 %, and no response in 30 ± 5 %. In Ca(2+)-free external medium, SCFs ceased completely and muscimol failed to elicit [Ca(2+)]i rises. All muscimol-induced [Ca(2+)]i changes were blocked by the GABAA-R antagonist bicuculline, suggesting that they result from changes in membrane potential depending on the cell's Cl(-) equilibrium potential. Unexpectedly, bicuculline increased the amplitude and frequency of SCFs in 54 % of CCs, revealing a tonic inhibition of SCFs by ambient GABA acting through GABAA-R. Mecamylamine (a specific nicotinic cholinergic blocker) decreased basal SCF activity in 18 % of CCs and inhibited bicuculline-induced SCF intensification, suggesting that spontaneous acetylcholine (ACh) release from nerve endings contributes to SCF generation in CCs in situ and that blockade of presynaptic GABAA-Rs intensifies SCFs in part through the disinhibition of spontaneous cholinergic transmission. Electrophysiological experiments confirmed that spontaneous excitatory postsynaptic currents recorded from CCs in situ were enhanced by bicuculline. To our knowledge, this is the first description of a regulatory effect of endogenous GABA on synaptic currents and SCFs of adrenal CCs. These findings denote a novel GABA-mediated presynaptic and postsynaptic regulatory mechanism of CC activity which may participate in the control of catecholamine secretion. PMID:26490458

  10. Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Agonist, GLP-1-IgG2σ-Fc.

    PubMed

    Yang, Yi; Chen, Fang; Wan, Deyou; Liu, Yunhui; Yang, Li; Feng, Hongru; Cui, Xinling; Gao, Xin; Song, Haifeng

    2016-01-01

    Human GLP-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes. However, its clinical benefits are limited by its short half-life, which is less than 2 min because of its small size and rapid enzymatic inactivation by dipeptidyl peptidase IV. We engineered GLP-1-IgG2σ-Fc, a 68-kDa fusion protein linking a variant human GLP-1 (A8G/G26E/R36G) to a human IgG2σ constant heavy-chain. A stably transfected Chinese hamster ovary cell line was obtained using electroporation. Western blotting showed that the expressed protein was immunoreactive to both GLP-1 and IgG antibodies. GLP-1-IgG2σ-Fc stimulated insulin secretion from INS-1 cells in a dose- and glucose-dependent manner and increased insulin mRNA expression. The half-life of GLP-1-IgG2σ-Fc in cynomolgus monkeys was approximately 57.1 ± 4.5 h. In the KKAy mouse model of diabetes, one intraperitoneal injection of GLP-1-IgG2σ-Fc (1 mg/kg) reduced blood glucose levels for 5 days. A 4-week repeat-administration study identified sustained effects on blood glucose levels. Oral glucose tolerance tests conducted at the beginning and end of this 4-week period showed that GLP-1-IgG2σ-Fc produced a stable glucose lowering effect. In addition, KKAy mice treated with GLP-1-IgG2σ-Fc showed statistically significant weight loss from day 23. In conclusion, these properties of GLP-1-IgG2σ-Fc demonstrated that it represented a potential long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes. PMID:27232339

  11. Modulation of silent and constitutively active nociceptin/orphanin FQ receptors by potent receptor antagonists and Na+ ions in rat sympathetic neurons.

    PubMed

    Mahmoud, Saifeldin; Margas, Wojciech; Trapella, Claudio; Caló, Girolamo; Ruiz-Velasco, Victor

    2010-05-01

    pharmacology and allosteric regulation by Na(+). Data are also presented demonstrating that heterologously expressed mu opioid receptors in sympathetic neurons are similarly modulated. PMID:20159949

  12. Dual effect of trimebutine on contractility of the guinea pig ileum via the opioid receptors.

    PubMed

    Taniyama, K; Sano, I; Nakayama, S; Matsuyama, S; Takeda, K; Yoshihara, C; Tanaka, C

    1991-12-01

    Preparations of longitudinal muscle attached to myenteric plexus from guinea pig ileum were used to observe the effect of trimebutine on intestinal motility. Electrical stimulation at 0.2 Hz and 5 Hz produced contraction mediated by the release of acetylcholine in the preparations. The response to low-frequency stimulation (0.2 Hz) was inhibited by trimebutine (10(-8)-10(-5) mol/L), and the response to high-frequency stimulation (5 Hz) was enhanced by the drug at low concentrations (10(-8)-10(-7) mol/L) and inhibited by high concentrations (10(-6)-10(-5) mol/L). This enhancement was mimicked by [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, and was antagonized by naloxone but not by MR2266. Enhancement by trimebutine was inhibited by yohimbine. Trimebutine (greater than or equal to 10(-8) mol/L) inhibited stimulation (5 Hz)-evoked release of norepinephrine, and the trimebutine effect was antagonized by naloxone but not by MR2266. Low concentrations of trimebutine inhibit norepinephrine release via the mu-opioid receptor and enhance intestinal motility by preventing the adrenergic inhibition of acetylcholine release. Inhibition by trimebutine was antagonized either by naloxone or MR2266. High concentrations of trimebutine may inhibit acetylcholine release via the mu- and kappa-opioid receptors, after which the intestinal motility is inhibited. Trimebutine at further high concentrations (greater than 10(-5) mol/L) contracted single smooth muscle cells from the circular muscle layers but not from the longitudinal muscle layers. The usual dose of trimebutine may exert dual effect on the intestinal motility indirectly through cholinergic and adrenergic neurons without direct effect on the smooth muscle. PMID:1659547

  13. A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-11-27

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs. PMID:26432642

  14. Cytokine-Induced Loss of Glucocorticoid Function: Effect of Kinase Inhibitors, Long-Acting β2-Adrenoceptor Agonist and Glucocorticoid Receptor Ligands

    PubMed Central

    Rider, Christopher F.; Shah, Suharsh; Miller-Larsson, Anna; Giembycz, Mark A.; Newton, Robert

    2015-01-01

    Acting on the glucocorticoid receptor (NR3C1), glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2×glucocorticoid response element (GRE) reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF) or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h) was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK) inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3 expression by TNF. Finally

  15. Autoradiographic comparison of the distribution of the neutral endopeptidase enkephalinase and of. mu. and delta opioid receptors in rat brain

    SciTech Connect

    Waksman, G.; Hamel, E.; Fournie-Zaluski, M.C.; Roques, B.P.

    1986-03-01

    The neutral endopeptidase EC 3.4.24.11, also designated enkephalinase, has been visualized by in vitro autoradiography using the tritiated inhibitor (/sup 3/H)-N-((2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)glycine, ((/sup 3/H)HACBO-Gly). Specific binding of (/sup 3/H)HACBO-Gly corresponding to 85% of the total binding to brain slices was inhibited by 1 ..mu..M thiorphan, a selective inhibitor of enkephalinase, but remained unchanged in the presence of captopril, a selective inhibitor of angiotensin-converting enzyme. Very high levels of (/sup 3/H)HACBO-Gly binding were found in the choroid plexus and the substantia nigra. High levels were present in the caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercle, and in the substantia gelatinosa of the spinal cord. The distribution of enkephalinase was compared to that of ..mu.. and delta opioid receptors, selectively labeled with (/sup 3/H)Tyr-D-Ala-Gly-MePhe-glycinol and (/sup 3/H)Try-D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate putamen, (/sup 3/H)HACBO-Gly binding overlapped the clustered ..mu.. sites but appeared more closely related to the diffusely distributed delta sites. The association of enkephalinase with delta and ..mu.. opioid receptors in these areas is consistent with the observed role of the enzyme in regulating the effects of opioid peptides in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in numerous rat brain areas between the distribution of enkephalinase and that of ..mu.. and/ or delta opioid binding sites could account for most of the pharmacological effects elicited by enkephalinase inhibitors.

  16. Characterization of kappa opioid receptor mediated, dynorphin-stimulated [35S]GTPγS binding in mouse striatum for the evaluation of selective KOR ligands in an endogenous setting.

    PubMed

    Zhou, Lei; Stahl, Edward L; Lovell, Kimberly M; Frankowski, Kevin J; Prisinzano, Thomas E; Aubé, Jeffrey; Bohn, Laura M

    2015-12-01

    Differential modulation of kappa opioid receptor (KOR) signaling has been a proposed strategy for developing therapies for drug addiction and depression by either activating or blocking this receptor. Hence, there have been significant efforts to generate ligands with diverse pharmacological properties including partial agonists, antagonists, allosteric modulators as well as ligands that selectively activate some pathways while not engaging others (biased agonists). It is becoming increasingly evident that G protein coupled receptor signaling events are context dependent and that what may occur in cell based assays may not be fully indicative of signaling events that occur in the naturally occurring environment. As new ligands are developed, it is important to assess their signaling capacity in relevant endogenous systems in comparison to the performance of endogenous agonists. Since KOR is considered the cognate receptor for dynorphin peptides we have evaluated the selectivity profiles of dynorphin peptides in wild-type (WT), KOR knockout (KOR-KO), and mu opioid receptor knockout (MOR-KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that while the small molecule KOR agonist U69,593, is very selective for KOR, dynorphin peptides promiscuously stimulate G protein signaling in striatum. Furthermore, our studies demonstrate that norBNI and 5'GNTI are highly nonselective antagonists as they maintain full potency and efficacy against dynorphin signaling in the absence of KOR. Characterization of a new KOR antagonist, which may be more selective than NorBNI and 5'GNTI, is presented using this approach. PMID:26160155

  17. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum.

    PubMed

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J; Mikami, Dean J; Wood, Jackie D

    2015-06-01

    Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

  18. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum

    PubMed Central

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J.; Mikami, Dean J.

    2015-01-01

    Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

  19. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    PubMed

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women. PMID:26713784

  20. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    SciTech Connect

    Zhang, Bing; Liu, Caini; Qian, Wen; Han, Yue; Li, Xiaoxia; Deng, Junpeng

    2014-05-01

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

  1. Dopamine D2 Receptors Act Upstream of AVP in the Latero-Anterior Hypothalamus to Modulate Adolescent Anabolic/Androgenic Steroid-Induced Aggression in Syrian Hamsters

    PubMed Central

    Morrison, Thomas R.; Ricci, Lesley A.; Melloni, Richard H.

    2015-01-01

    In pubertal male Syrian hamsters, exposure to anabolic/androgenic steroids (AAS) during adolescence facilitates a high level of offensive aggression modulated by the enhanced development and activity of the vasopressin (AVP) and dopamine (DA) neural systems within the latero-anterior hypothalamus (LAH), i.e., a brain region implicated in the control of aggression. The present studies provide a detailed report of the pharmacologic interactions between AVP and DA D2 receptor signaling within the LAH in the control of adolescent AAS-induced offensive aggression. Male Syrian hamsters were treated with AAS throughout adolescence and tested for aggression after local infusion of the DA D2 receptor antagonist eticlopride (ETIC) alone, or in combination with AVP in the LAH in an effort to determine the influence of DA D2 receptors relative to AVP-receptor mediated aggression mechanisms. As previously shown, ETIC infusion into the LAH suppressed adolescent AAS-induced aggressive responding; however, the AAS-induced aggressive phenotype was rescued by the co-infusion of AVP into the LAH. These behavioral data indicate that interactions between AVP and DA neural systems within the LAH modulate the control of aggression following adolescent exposure to AAS and that DA D2 receptor signaling functions upstream of AVP in the LAH to control this behavioral response. PMID:25798632

  2. Effects of stress and. beta. -funal trexamine pretreatment on morphine analgesia and opioid binding in rats

    SciTech Connect

    Adams, J.U.; Andrews, J.S.; Hiller, J.M.; Simon, E.J.; Holtzman, S.G.

    1987-12-28

    This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opiods which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 yr or unstressed were injected ICV with either saline or 2.5 ..mu..g of ..beta..-funaltrexamine (..beta..-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia or were sacrificed and opioid binding in brain was determined. (/sup 3/H)D-Ala/sup 2/NMePhe/sup 4/-Gly/sup 5/(ol)enkephalin (DAGO) served as a specific ligand for mu-opioid receptors, and (/sup 3/H)-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. ..beta..-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with ..beta..-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received ..beta..-FNA while unstressed. ..beta..-FNA caused small and similar decreases in (/sup 3/H)-DAGO binding in brain of both stressed and unstressed animals. 35 references, 2 figures, 2 tables.

  3. Orphan nuclear receptor NR2F6 acts as an essential gatekeeper of Th17 CD4+ T cell effector functions

    PubMed Central

    2014-01-01

    Members of the evolutionarily conserved family of the chicken ovalbumin upstream promoter transcription factor NR2F/COUP-TF orphan receptors have been implicated in lymphocyte biology, ranging from activation to differentiation and elicitation of immune effector functions. In particular, a CD4+ T cell intrinsic and non-redundant function of NR2F6 as a potent and selective repressor of the transcription of the pro-inflammatory cytokines interleukin (Il) 2, interferon y (ifng) and consequently of T helper (Th)17 CD4+ T cell-mediated autoimmune disorders has been discovered. NR2F6 serves as an antigen receptor signaling threshold-regulated barrier against autoimmunity where NR2F6 is part of a negative feedback loop that limits inflammatory tissue damage induced by weakly immunogenic antigens such as self-antigens. Under such low affinity antigen receptor stimulation, NR2F6 appears as a prototypical repressor that functions to “lock out” harmful Th17 lineage effector transcription. Mechanistically, only sustained high affinity antigen receptor-induced protein kinase C (PKC)-mediated phosphorylation has been shown to inactivate NR2F6, thereby displacing pre-bound NR2F6 from the DNA and, subsequently, allowing for robust NFAT/AP-1- and RORγt-mediated cytokine transcription. The NR2F6 target gene repertoire thus identifies a general anti-inflammatory gatekeeper role for this orphan receptor. Investigating these signaling pathway(s) will enable a greater knowledge of the genetic, immune, and environmental mechanisms that lead to chronic inflammation and of certain autoimmune disorders in a given individual. PMID:24919548

  4. Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using (/sup 125/I)FK33824

    SciTech Connect

    Rothman, R.B.; Jacobson, A.E.; Rice, K.C.; Herkenham, M.

    1987-11-01

    Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites (25,35), consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, (/sup 3/H)oxymorphone, (/sup 3/H)D-ala2-MePhe4, Gly-ol5-enkephalin (DAGO), and (/sup 125/I)D-ala2-Me-Phe4-met(o)-ol)enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu binding sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand (/sup 125/I)FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.

  5. Synthesis of novel cognition enhancers with pyrazolo[5,1-c][1,2,4]benzotriazine core acting at γ-aminobutyric acid type A (GABA(A)) receptor.

    PubMed

    Guerrini, Gabriella; Ciciani, Giovanna; Costanzo, Annarella; Daniele, Simona; Martini, Claudia; Ghelardini, Carla; Di Cesare Mannelli, Lorenzo; Ciattini, Samuele

    2013-04-15

    Memory dysfunction associated with aging, neurodegenerative and psychiatric disorders represents an increasing medical need. Advances in research exploring the biological mechanisms underlying learning and memory have opened new potential approaches for development of memory-enhancing therapies addressed to selective neuronal targets. In this work, we synthesized some derivatives with a pyrazolo[5,1-c][1,2,4]benzotriazine core to identify ligands on GABAA receptors subtype (benzodiazepine site on GABAA-receptor) endowed with the potential of enhancing cognition activity without the side effects usually associated with non-selective GABAA modulators. In fact, there is much evidence that GABAA-R (γ-aminobutyric acid, type A receptor) subtype ligands have relevance in learning and memory. In vitro and in vivo tests have been performed. Pharmacological data indicate that compounds 7, 13, 14 and 22 act as dual functional modulators of GABAA-Rs (promnemonic and anxiolytic agents) while only compounds 3 and 10 stand out as selectively displaying good antiamnesic and procognitive activity (1 and 3 mg/kg, respectively). PMID:23490154

  6. 2,4,5-Trimethylimidazolium Scaffold for Anion Recognition Receptors Acting Through Charge-Assisted Aliphatic and Aromatic C-H Interactions.

    PubMed

    Sabater, Paula; Zapata, Fabiola; Caballero, Antonio; Fernández, Israel; Ramirez de Arellano, Carmen; Molina, Pedro

    2016-05-01

    A series of two-armed 2,4,5-trimethylimidazolium-based oxoanion receptors, which incorporate two end-capped photoactive anthracene rings, being the central core an aromatic or heteroaromatic ring, has been designed. In the presence of HP2O7(3-), H2PO4(-), and SO4(2-) anions, (1)H- and (31)P NMR spectroscopical data clearly indicate the simultaneous occurrence of several charge-assisted aliphatic and aromatic C-H noncovalent interactions, i.e., significant downfield shifts were observed for the imidazolium C(2)-CH3 protons, the methylene N-CH2 protons, and the inner aromatic proton or the outer heteroaromatic protons. Density functional theory calculations confirm the occurrence of these noncovalent interaction and suggest that the interaction between the anions and the receptors is mainly electrostatic in nature. PMID:27078523

  7. Acting on Hormone Receptors with Minimal Side Effect on Cell Proliferation: A Timely Challenge Illustrated with GLP-1R and GPER

    PubMed Central

    Gigoux, Véronique; Fourmy, Daniel

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute a large family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and cellular responses. GPCR are involved in a wide variety of physiological processes, including in the neuroendocrine system. GPCR are also involved in many diseases and are the target of 30% of marketed medicinal drugs. Whereas the majority of the GPCR-targeting drugs have proved their therapeutic benefit, some of them were associated with undesired effects. We develop two examples of used drugs whose therapeutic benefits are tarnished by carcinogenesis risks. The chronic administration of glucagon-like peptide-1 (GLP-1) analogs widely used to treat type-2 diabetes was associated with an increased risk of pancreatic or thyroid cancers. The long-term treatment with the estrogen antagonist tamoxifen, developed to target breast cancer overexpressing estrogen receptors ER, presents agonist activity on the G protein-coupled estrogen receptor which is associated with an increased incidence of endometrial cancer and breast cancer resistance to hormonotherapy. We point out and discuss the need of pharmacological studies to understand and overcome the undesired effects associated with the chronic administration of GPCR ligands. In fact, biological effects triggered by GPCR often result from the activation of multiple intracellular signaling pathways. Deciphering which signaling networks are engaged following GPCR activation appears to be primordial to unveil their contribution in the physiological and physiopathological processes. The development of biased agonists to elucidate the role of the different signaling mechanisms mediated by GPCR activation will allow the generation of new therapeutic agents with improved efficacy and reduced side effects. In this regard, the identification of GLP-1R biased ligands promoting insulin secretion without inducing pro-tumoral effects would offer therapeutic benefit. PMID

  8. Regional distribution of serotonergic receptors: a systems neuroscience perspective on the downstream effects of the multimodal-acting antidepressant vortioxetine on excitatory and inhibitory neurotransmission.

    PubMed

    Pehrson, Alan L; Jeyarajah, Theepica; Sanchez, Connie

    2016-04-01

    Previous work from this laboratory hypothesized that the multimodal antidepressant vortioxetine enhances cognitive function through a complex mechanism, using serotonergic (5-hydroxytryptamine, 5-HT) receptor actions to modulate gamma-butyric acid (GABA) and glutamate neurotransmission in key brain regions like the prefrontal cortex (PFC) and hippocampus. However, serotonergic receptors have circumscribed expression patterns, and therefore vortioxetine's effects on GABA and glutamate neurotransmission will probably be regionally selective. In this article, we attempt to develop a conceptual framework in which the effects of 5-HT, selective serotonin reuptake inhibitors (SSRIs), and vortioxetine on GABA and glutamate neurotransmission can be understood in the PFC and striatum-2 regions with roles in cognition and substantially different 5-HT receptor expression patterns. Thus, we review the anatomy of the neuronal microcircuitry in the PFC and striatum, anatomical data on 5-HT receptor expression within these microcircuits, and electrophysiological evidence on the effects of 5-HT on the behavior of each cell type. This analysis suggests that 5-HT and SSRIs will have markedly different effects within the PFC, where they will induce mixed effects on GABA and glutamate neurotransmission, compared to the striatum, where they will enhance GABAergic interneuron activity and drive down the activity of medium spiny neurons. Vortioxetine is expected to reduce GABAergic interneuron activity in the PFC and concomitantly increase cortical pyramidal neuron firing. However in the striatum, vortioxetine is expected to increase activity at GABAergic interneurons and have mixed excitatory and inhibitory effects in medium spiny neurons. Thus the conceptual framework developed here suggests that vortioxetine will have regionally selective effects on GABA and glutamate neurotransmission. PMID:26250622

  9. Cannabidiol induces rapid-acting antidepressant-like effects and enhances cortical 5-HT/glutamate neurotransmission: role of 5-HT1A receptors.

    PubMed

    Linge, Raquel; Jiménez-Sánchez, Laura; Campa, Leticia; Pilar-Cuéllar, Fuencisla; Vidal, Rebeca; Pazos, Angel; Adell, Albert; Díaz, Álvaro

    2016-04-01

    Cannabidiol (CBD), the main non-psychotomimetic component of marihuana, exhibits anxiolytic-like properties in many behavioural tests, although its potential for treating major depression has been poorly explored. Moreover, the mechanism of action of CBD remains unclear. Herein, we have evaluated the effects of CBD following acute and chronic administration in the olfactory bulbectomy mouse model of depression (OBX), and investigated the underlying mechanism. For this purpose, we conducted behavioural (open field and sucrose preference tests) and neurochemical (microdialysis and autoradiography of 5-HT1A receptor functionality) studies following treatment with CBD. We also assayed the pharmacological antagonism of the effects of CBD to dissect out the mechanism of action. Our results demonstrate that CBD exerts fast and maintained antidepressant-like effects as evidenced by the reversal of the OBX-induced hyperactivity and anhedonia. In vivo microdialysis revealed that the administration of CBD significantly enhanced serotonin and glutamate levels in vmPFCx in a different manner depending on the emotional state and the duration of the treatment. The potentiating effect upon neurotransmitters levels occurring immediately after the first injection of CBD might underlie the fast antidepressant-like actions in OBX mice. Both antidepressant-like effect and enhanced cortical 5-HT/glutamate neurotransmission induced by CBD were prevented by 5-HT1A receptor blockade. Moreover, adaptive changes in pre- and post-synaptic 5-HT1A receptor functionality were also found after chronic CBD. In conclusion, our findings indicate that CBD could represent a novel fast antidepressant drug, via enhancing both serotonergic and glutamate cortical signalling through a 5-HT1A receptor-dependent mechanism. PMID:26711860

  10. A steroid receptor coactivator acts as the DNA-binding partner of the methoprene-tolerant protein in regulating juvenile hormone response genes.

    PubMed

    Li, Meng; Liu, Pengcheng; Wiley, Jessica D; Ojani, Reyhaneh; Bevan, David R; Li, Jianyong; Zhu, Jinsong

    2014-08-25

    Methoprene-tolerant (Met) protein is a juvenile hormone (JH) receptor in insects. JH-bound Met forms a complex with the βFtz-F1-interacting steroid receptor coactivator (FISC) and together they regulate JH response genes in mosquitoes. Both proteins contain basic helix-loop-helix (bHLH) and PAS motifs. Here we demonstrated that FISC is the obligatory partner of Met for binding to JH-response elements (JHREs). Met or FISC alone could not bind a previously characterized JHRE, while formation of the Met-FISC complex was necessary and sufficient to bind to the JHRE. This binding required participation of the DNA-binding domains of both Met and FISC. The optimal DNA sequence recognized by Met and FISC contained a core consensus sequence GCACGTG. While formation of the Met-FISC complex in mosquito cells was induced by JH, heterodimerization and DNA binding of bacterially expressed Met and FISC were JH-independent, implying that additional mosquito proteins were required to modulate formation of the receptor complex. PMID:25004255

  11. Structure Activity Relationship of (N)-Methanocarba Phosphonate Analogues of 5’-AMP as Cardioprotective Agents Acting Through a Cardiac P2X Receptor

    PubMed Central

    Kumar, T. Santhosh; Zhou, Si-Yuan; Joshi, Bhalchandra V.; Balasubramanian, Ramachandran; Yang, Tiehong; Liang, Bruce T.; Jacobson, Kenneth A.

    2010-01-01

    P2X receptor activation protects in heart failure models. MRS2339 3, a 2-chloro-AMP derivative containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system, activates this cardioprotective channel. Michaelis–Arbuzov and Wittig reactions provided phosphonate analogues of 3, expected to be stable in vivo due to the C-P bond. After chronic administration via a mini-osmotic pump (Alzet), some analogues significantly increased intact heart contractile function in calsequestrin-overexpressing mice (genetic model of heart failure) compared to vehicle-infused mice (all inactive at the vasodilatory P2Y1 receptor). Two phosphonates, (1’S,2’R,3’S,4’R,5’S)-4’-(6-amino-2-chloropurin-9-yl)-2’,3’-(dihydroxy)-1’-(phosphonomethylene)-bicyclo[3.1.0]hexane 4 and its homologue 9, both 5’-saturated, containing a 2-Cl substitution, improved echocardiography-derived fractional shortening (20.25% and 19.26%, respectively, versus 13.78% in controls), while unsaturated 5’-extended phosphonates, all 2-H analogues, and a CH3-phosphonate were inactive. Thus, chronic administration of nucleotidase-resistant phosphonates conferred a beneficial effect, likely via cardiac P2X receptor activation. Thus, we have greatly expanded the range of carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure. PMID:20192270

  12. A Steroid Receptor Coactivator Acts as the DNA-binding Partner of the Methoprene-tolerant Protein in Regulating Juvenile Hormone Response Genes

    PubMed Central

    Li, Meng; Liu, Pengcheng; Wiley, Jessica D.; Ojani, Reyhaneh; Bevan, David R.; Li, Jianyong; Zhu, Jinsong

    2014-01-01

    Methoprene-tolerant (Met) protein is a juvenile hormone (JH) receptor in insects. JH-bound Met forms a complex with the βFtz-F1-interacting steroid receptor coactivator (FISC) and together they regulate JH response genes in mosquitoes. Both proteins contain basic-helix-loop-helix (bHLH) and PAS motifs. Here we demonstrated that FISC is the obligatory partner of Met for binding to JH-response elements (JHREs). Met or FISC alone could not bind a previously characterized JHRE, while formation of the Met-FISC complex was necessary and sufficient to bind to the JHRE. This binding required participation of the DNA-binding domains of both Met and FISC. The optimal DNA sequence recognized by Met and FISC contained a core consensus sequence GCACGTG. While formation of the Met-FISC complex in mosquito cells was induced by JH, heterodimerization and DNA binding of bacterially expressed Met and FISC were JH-independent, implying that additional mosquito proteins were required to modulate formation of the receptor complex. PMID:25004255

  13. Cannabinoid-1 (CB1) receptors regulate colonic propulsion by acting at motor neurons within the ascending motor pathways in mouse colon.

    PubMed

    Sibaev, Andrei; Yüce, Birol; Kemmer, Markus; Van Nassauw, Luc; Broedl, Ulli; Allescher, Hans D; Göke, Burkhard; Timmermans, Jean-Pierre; Storr, Martin

    2009-01-01

    Cannabinoid-1 (CB(1)) receptors on myenteric neurons are involved in the regulation of intestinal motility. Our aim was to investigate CB(1) receptor involvement in ascending neurotransmission in mouse colon and to characterize the involved structures by functional and morphological means. Presence of the CB(1) receptor was investigated by RT-PCR, and immunohistochemistry was used for colabeling studies. Myenteric reflex responses were initiated by electrical stimulation (ES) at different distances, and junction potentials (JP) were recorded from circular smooth muscle cells by intracellular recording in an unpartitioned and a partitioned recording chamber. In vivo colonic propulsion was tested in wild-type and CB(1)(-/-) mice. Immunostaining with the cytoskeletal marker peripherin showed CB(1) immunoreactivity both on Dogiel type I and type II neurons. Further neurochemical characterization revealed CB(1) on choline acetyltransferase-, calretinin-, and 5-HT-immunopositive myenteric neurons, but nitrergic neurons appeared immunonegative for CB(1) immunostaining. Solitary spindle-shaped CB(1)-immunoreactive cells in between smooth muscle cells lacked specific markers for interstitial cells of Cajal or glial cells. ES elicited neuronally mediated excitatory JP (EJP) and inhibitory JP. Gradual increases in distance resulted in a wave-like EJP with EJP amplitudes being maximal at the location of stimulating electrode 6 and a maximal EJP projection distance of approximately 18 mm. The CB(1) receptor agonist WIN 55,212-2 reduced the amplitude of EJP and was responsible for shortening the oral spreading of the excitatory impulse. In a partitioned chamber, WIN 55,212-2 reduced EJP at the separated oral sites, proving that CB(1) activation inhibits interneuron-mediated neurotransmission. These effects were absent in the presence of the CB(1) antagonist SR141716A, which, when given alone, had no effect. WIN 55,212-2 inhibited colonic propulsion in wild-type mice but not in

  14. Inflammatory Pain Promotes Increased Opioid Self-Administration: Role of Dysregulated Ventral Tegmental Area μ Opioid Receptors

    PubMed Central

    Hipólito, Lucia; Wilson-Poe, Adrianne; Campos-Jurado, Yolanda; Zhong, Elaine; Gonzalez-Romero, Jose; Virag, Laszlo; Whittington, Robert; Comer, Sandra D.; Carlton, Susan M.; Walker, Brendan M.; Bruchas, Michael R.

    2015-01-01

    Pain management in opioid abusers engenders ethical and practical difficulties for clinicians, often resulting in pain mismanagement. Although chronic opioid administration may alter pain states, the presence of pain itself may alter the propensity to self-administer opioids, and previous history of drug abuse comorbid with chronic pain promotes higher rates of opioid misuse. Here, we tested the hypothesis that inflammatory pain leads to increased heroin self-administration resulting from altered mu opioid receptor (MOR) regulation of mesolimbic dopamine (DA) transmission. To this end, the complete Freund's adjuvant (CFA) model of inflammation was used to assess the neurochemical and functional changes induced by inflammatory pain on MOR-mediated mesolimbic DA transmission and on rat intravenous heroin self-administration under fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement. In the presence of inflammatory pain, heroin intake under an FR schedule was increased for high, but attenuated for low, heroin doses with concomitant alterations in mesolimbic MOR function suggested by DA microdialysis. Consistent with the reduction in low dose FR heroin self-administration, inflammatory pain reduced motivation for a low dose of heroin, as measured by responding under a PR schedule of reinforcement, an effect dissociable from high heroin dose PR responding. Together, these results identify a connection between inflammatory pain and loss of MOR function in the mesolimbic dopaminergic pathway that increases intake of high doses of heroin. These findings suggest that pain-induced loss of MOR function in the mesolimbic pathway may promote opioid dose escalation and contribute to opioid abuse-associated phenotypes. SIGNIFICANCE STATEMENT This study provides critical new insights that show that inflammatory pain alters heroin intake through a desensitization of MORs located within the VTA. These findings expand our knowledge of the interactions between

  15. Involvement of nitric oxide in the inhibition of bone cancer-induced hyperalgesia through the activation of peripheral opioid receptors in mice.

    PubMed

    Menéndez, Luis; Juárez, Lucía; García, Verónica; Hidalgo, Agustín; Baamonde, Ana

    2007-07-01

    Experiments were designed to elucidate the involvement of nitric oxide (NO) in the antihyperalgesic effect induced by the activation of peripheral mu-opioid receptors on osteosarcoma-induced thermal hyperalgesia in mice. Since this pathway has previously been shown to be involved in the antihyperalgesic effect induced by some drugs--including opiates--on inflammatory pain, experiments were also performed in inflamed mice. The intraplantar administration of loperamide (15 microg) abolishes the thermal hyperalgesia that appears 4 weeks after the intratibial inoculation of NCTC 2472 cells in C3H/HeJ mice. The blockade of this effect by coadministering a peripheral opioid receptor antagonist (naloxone methiodide), a nitric oxide synthase (NOS) inhibitor (L-NMMA), a soluble guanylyl cyclase inhibitor (ODQ), a PKG inhibitor (KT-5823) or a K(+)(ATP)-channel blocker (glibenclamide) shows the involvement of a NO/cGMP/K(+)(ATP)-channel pathway. Accordingly the administration of loperamide produced, in osteosarcoma-bearing mice, an increase in the concentrations of NO metabolites, nitrites and nitrates, extracted from paws. The selective inhibitor of eNOS L-NIO, but not the inhibitors of nNOS (N-omega-propyl-L-arginine) or iNOS (1400w), blocked the effect of loperamide on osteosarcoma-induced hyperalgesia and also the endogenous opioid peripheral hypoalgesia that appears during the initial stages of the development of this osteosarcoma. Although this pathway also participates in the inhibitory effect of loperamide on the thermal hyperalgesia induced by administration of complete Freund's adjuvant, only selective inhibitors of nNOS or iNOS antagonized this effect. Our results demonstrate that the activation of a NO/cGMP/K(+)(ATP)-channel triggered by eNOS participates in the peripheral antihyperalgesic of loperamide on osteosarcoma-induced thermal hyperalgesia. PMID:17543351

  16. Prenatal exposure to vanilla or alcohol induces crawling after these odors in the neonate rat: The role of mu and kappa opioid receptor systems.

    PubMed

    Gaztañaga, Mirari; Aranda-Fernández, P Ezequiel; Chotro, M Gabriela

    2015-09-01

    Rat fetuses can perceive chemosensory stimuli derived from their mother's diet, and they may learn about those stimuli. In previous studies we have observed that prenatal exposure to alcohol during the last days of gestation increases the acceptance and liking of an alcohol flavor in infant and adolescent rats. While these results were not found after prenatal exposure to vanilla, cineole or anise, suggesting that the pharmacological properties of alcohol, mediated by the opioid system, underlie the effects observed with this drug. Considering that other studies report enhanced acceptance of non-alcohol flavors experienced prenatally when subjects were tested before infancy, we explore the possibility of observing similar results if testing 1-day old rats exposed prenatally to vanilla. Using an "odor-induced crawling" testing procedure, it was observed that neonates exposed prenatally to vanilla or alcohol crawl for a longer distance towards the experienced odor than to other odors or than control pups. Blocking mu, but not kappa opioid receptors, reduced the attraction of vanilla odor to neonates exposed to vanilla in utero, while the response to alcohol in pups exposed prenatally to this drug was affected by both antagonists. Results confirm that exposure to a non-alcohol odor enhances postnatal responses to it, observable soon after birth, while also suggesting that the mu opioid receptor system plays an important role in generating this effect. The results also imply that with alcohol exposure, the prenatal opioid system is wholly involved, which could explain the longer retention of the enhanced attraction to alcohol following prenatal experience with the drug. PMID:25554482

  17. Discovery of the first small-molecule opioid pan antagonist with nanomolar affinity at mu, delta, kappa, and nociceptin opioid receptors.

    PubMed

    Zaveri, Nurulain T; Journigan, V Blair; Polgar, Willma E

    2015-04-15

    The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [(35)S]GTPγS functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure-activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [(35)S]GTPγS assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3-6-fold more potent than those of JDTic. AT-076 is the

  18. Discovery of the First Small-Molecule Opioid Pan Antagonist with Nanomolar Affinity at Mu, Delta, Kappa, and Nociceptin Opioid Receptors

    PubMed Central

    2016-01-01

    The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [35S]GTPγS functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure–activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [35S]GTPγS assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3–6-fold more potent than those of JDTic. AT-076 is the

  19. Glycyrrhetinic acid and E.resveratroloside act as potential plant derived compounds against dopamine receptor D3 for Parkinson's disease: a pharmacoinformatics study.

    PubMed

    Mirza, Muhammad Usman; Mirza, A Hammad; Ghori, Noor-Ul-Huda; Ferdous, Saba

    2015-01-01

    Parkinson's disease (PD) is caused by loss in nigrostriatal dopaminergic neurons and is ranked as the second most common neurodegenerative disorder. Dopamine receptor D3 is considered as a potential target in drug development against PD because of its lesser side effects and higher degree of neuro-protection. One of the prominent therapies currently available for PD is the use of dopamine agonists which mimic the natural action of dopamine in the brain and stimulate dopamine receptors directly. Unfortunately, use of these pharmacological therapies such as bromocriptine, apomorphine, and ropinirole provides only temporary relief of the disease symptoms and is frequently linked with insomnia, anxiety, depression, and agitation. Thus, there is a need for an alternative treatment that not only hinders neurodegeneration, but also has few or no side effects. Since the past decade, much attention has been given to exploitation of phytochemicals and their use in alternative medicine research. This is because plants are a cheap, indispensable, and never ending resource of active compounds that are beneficial against various diseases. In the current study, 40 active phytochemicals against PD were selected through literature survey. These ligands were docked with dopamine receptor D3 using AutoDock and AutoDockVina. Binding energies were compared to docking results of drugs approved by the US Food and Drug Administration against PD. The compounds were further analyzed for their absorption, distribution, metabolism, and excretion-toxicity profile. From the study it is concluded that glycyrrhetinic acid and E.resveratroloside are potent compounds having high binding energies which should be considered as potential lead compounds for drug development against PD. PMID:25565772

  20. Timosaponin derivative YY-23 acts as a non-competitive NMDA receptor antagonist and exerts a rapid antidepressant-like effect in mice

    PubMed Central

    Zhang, Qi; Guo, Fei; Fu, Zhi-wen; Zhang, Bing; Huang, Cheng-gang; Li, Yang

    2016-01-01

    Aim: N-methyl-D-aspartic acid (NMDA) receptor modulators have shown promising results as potential antidepressant agents, whereas timosaponins extracted from the Chinese herb Rhizoma Anemarrhenae exhibit antidepressant activities. In the present study we examined whether YY-23, a modified metabolite of timosaponin B-III, could affect NMDA receptors in rat hippocampal neurons in vitro, and evaluated its antidepressant-like effects in stressed mice. Methods: NMDA-induced currents were recorded in acutely dissociated rat hippocampal CA1 neurons using a whole-cell recording technique. C57BL/6 mice were exposed to a 6-week chronic mild stress (CMS) or a 10-d chronic social defeat stress (CSDS). The stressed mice were treated with YY-23 (20 mg·kg−1·d−1) or a positive-control drug, fluoxetine (10 mg·kg−1·d−1) for 3 weeks. Behavioral assessments were carried out every week. Results: In acutely dissociated rat hippocampal CA1 neurons, YY-23 selectively and reversibly inhibited NMDA-induced currents with an EC50 value of 2.8 μmol/L. This inhibition of NMDA-induced currents by YY-23 was non-competitive, and had no features of voltage-dependency or use-dependency. Treatment of the stressed mice with YY-23 not only reversed CMS-induced deficiency of sucrose preference and immobility time, and CSDS-induced reduction of social interaction, but also had faster onset as compared to fluoxetine. Conclusion: YY-23 is a novel non-competitive antagonist of NMDA receptors with promising rapid antidepressant-like effects in mouse models of CMS and CSDS depression. PMID:26687936

  1. Glycyrrhetinic acid and E.resveratroloside act as potential plant derived compounds against dopamine receptor D3 for Parkinson’s disease: a pharmacoinformatics study

    PubMed Central

    Mirza, Muhammad Usman; Mirza, A Hammad; Ghori, Noor-Ul-Huda; Ferdous, Saba

    2015-01-01

    Parkinson’s disease (PD) is caused by loss in nigrostriatal dopaminergic neurons and is ranked as the second most common neurodegenerative disorder. Dopamine receptor D3 is considered as a potential target in drug development against PD because of its lesser side effects and higher degree of neuro-protection. One of the prominent therapies currently available for PD is the use of dopamine agonists which mimic the natural action of dopamine in the brain and stimulate dopamine receptors directly. Unfortunately, use of these pharmacological therapies such as bromocriptine, apomorphine, and ropinirole provides only temporary relief of the disease symptoms and is frequently linked with insomnia, anxiety, depression, and agitation. Thus, there is a need for an alternative treatment that not only hinders neurodegeneration, but also has few or no side effects. Since the past decade, much attention has been given to exploitation of phytochemicals and their use in alternative medicine research. This is because plants are a cheap, indispensable, and never ending resource of active compounds that are beneficial against various diseases. In the current study, 40 active phytochemicals against PD were selected through literature survey. These ligands were docked with dopamine receptor D3 using AutoDock and AutoDockVina. Binding energies were compared to docking results of drugs approved by the US Food and Drug Administration against PD. The compounds were further analyzed for their absorption, distribution, metabolism, and excretion-toxicity profile. From the study it is concluded that glycyrrhetinic acid and E.resveratroloside are potent compounds having high binding energies which should be considered as potential lead compounds for drug development against PD. PMID:25565772

  2. The Arf and Rab11 effector FIP3 acts synergistically with ASAP1 to direct Rabin8 in ciliary receptor targeting

    PubMed Central

    Wang, Jing; Deretic, Dusanka

    2015-01-01

    ABSTRACT Primary cilia have gained considerable importance in biology and disease now that their involvement in a wide range of human ciliopathies has been abundantly documented. However, detailed molecular mechanisms for specific targeting of sensory receptors to primary cilia are still unknown. Here, we show that the Arf and Rab11 effector FIP3 (also known as RAB11FIP3) promotes the activity of Rab11a and the Arf GTPase-activating protein (GAP) ASAP1 in the Arf4-dependent ciliary transport of the sensory receptor rhodopsin. During its passage out of the photoreceptor Golgi and trans-Golgi network (TGN), rhodopsin indirectly interacts with FIP3 through Rab11a and ASAP1. FIP3 competes with rhodopsin for binding to ASAP1 and displaces it from the ternary complex with Arf4–GTP and ASAP1. Resembling the phenotype resulting from lack of ASAP1, ablation of FIP3 abolishes ciliary targeting and causes rhodopsin mislocalization. FIP3 coordinates the interactions of ASAP1 and Rab11a with the Rab8 guanine nucleotide exchange factor Rabin8 (also known as RAB3IP). Our study implies that FIP3 functions as a crucial targeting regulator, which impinges on rhodopsin–ASAP1 interactions and shapes the binding pocket for Rabin8 within the ASAP1–Rab11a–FIP3 targeting complex, thus facilitating the orderly assembly and activation of the Rab11–Rabin8–Rab8 cascade during ciliary receptor trafficking. PMID:25673879

  3. Annexin A1 released from apoptotic cells acts through formyl peptide receptors to dampen inflammatory monocyte activation via JAK/STAT/SOCS signalling

    PubMed Central

    Pupjalis, Danute; Goetsch, Julia; Kottas, Diane J; Gerke, Volker; Rescher, Ursula

    2011-01-01

    The immunosuppressive effects of apoptotic cells involve inhibition of pro-inflammatory cytokine release and establishment of an anti-inflammatory cytokine profile, thus limiting the degree of inflammation and promoting resolution. We report here that this is in part mediated by the release of the anti-inflammatory mediator annexin A1 from apoptotic cells and the functional activation of annexin A1 receptors of the formyl peptide receptor (FPR) family on target cells. Supernatants from apoptotic neutrophils or the annexin A1 peptidomimetic Ac2-26 significantly reduced IL-6 signalling and the release of TNF-α from endotoxin-challenged monocytes. Ac2-26 activated STAT3 in a JAK-dependent manner, resulting in upregulated SOCS3 levels, and depletion of SOCS3 reversed the Ac2-26-mediated inhibition of IL-6 signalling. This identifies annexin A1 as part of the anti-inflammatory pattern of apoptotic cells and links the activation of FPRs to established signalling pathways triggering anti-inflammatory responses. PMID:21254404

  4. The anticonvulsant and behavioural profile of L-687,414, a partial agonist acting at the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex.

    PubMed

    Tricklebank, M D; Bristow, L J; Hutson, P H; Leeson, P D; Rowley, M; Saywell, K; Singh, L; Tattersall, F D; Thorn, L; Williams, B J

    1994-11-01

    1. The anticonvulsant and behavioural effects of the glycine/NMDA receptor partial agonist, L-687,414 (R(+)-cis-beta-methyl-3-amino-1-hydroxypyrrolid-2-one) have been investigated in rodents. 2. L-687,414 dose-dependently antagonized seizures induced by N-methyl-D,L- aspartic acid (NMDLA, ED50 = 19.7 mg kg-1), pentylenetetrazol (PTZ, ED50 = 13.0 mg kg-1) and electroshock (ED50 = 26.1 mg kg-1) when given intravenously 15 min before test, in male Swiss Webster mice but was most potent against audiogenic seizures induced by a 120 dB bell in DBA/2 mice (ED50 = 5.1 mg kg-1, i.p., 30 min before test). 3. L-687,414 also induced impairments of performance in a rotarod test in both Swiss Webster and DBA/2 mice and the ratio [rotarod MED:anticonvulsant ED50] varied between 0.9 and 5, depending on the convulsant used. 4. Similar behaviours to those seen after administration of the non-competitive NMDA receptor antagonist, MK-801 (head weaving, body rolling, hyperlocomotion) were seen in the mouse after giving L-687,414, although the peak effect occurred at a dose (100 mg kg-1) which was 5-20 times the anticonvulsant ED50S, depending on the convulsant used. Unlike MK-801, however, doses of L-687,414 that were behaviourally stimulant did not increase dopamine turnover in the nucleus accumbens. 5. Consistent with the interaction of L-687,414 with the glycine/NMDA receptor, the anticonvulsant, ataxic and motor stimulant effects of the compound were significantly attenuated by the glycine/NMDA receptor agonist, D-serine (10-100 micrograms per mouse, i.c.v.). 6. The results show that L-687,414 is a potent, orally active anticonvulsant with a more benign pharmacological profile than antagonists acting at the ion channel of the NMDA receptor complex. The compound is a useful tool with which to probe the functional role of the glycine co-agonist site in vivo. PMID:7858861

  5. Aromatic D-amino acids act as chemoattractant factors for human leukocytes through a G protein-coupled receptor, GPR109B.

    PubMed

    Irukayama-Tomobe, Yoko; Tanaka, Hirokazu; Yokomizo, Takehiko; Hashidate-Yoshida, Tomomi; Yanagisawa, Masashi; Sakurai, Takeshi

    2009-03-10

    GPR109B (HM74) is a putative G protein-coupled receptor (GPCR) whose cognate ligands have yet to be characterized. GPR109B shows a high degree of sequence similarity to GPR109A, another GPCR that was identified as a high-affinity nicotinic acid (niacin) receptor. However, the affinity of nicotinic acid to GPR109B is very low. In this study, we found that certain aromatic D-amino acids, including D-phenylalanine, D-tryptophan, and the metabolite of the latter, D-kynurenine, decreased the activity of adenylate cyclase in cells transfected with GPR109B cDNA through activation of pertussis toxin (PTX)-sensitive G proteins. These D-amino acids also elicited a transient rise of intracellular Ca(2+) level in cells expressing GPR109B in a PTX-sensitive manner. In contrast, these D-amino acids did not show any effects on cells expressing GPR109A. We found that the GPR109B mRNA is abundantly expressed in human neutrophils. D-phenylalanine and D-tryptophan induced a transient increase of intracellular Ca(2+) level and a reduction of cAMP levels in human neutrophils. Furthermore, knockdown of GPR109B by RNA interference inhibited the D-amino acids-induced decrease of cellular cAMP levels in human neutrophils. These D-amino acids induced chemotactic activity of freshly prepared human neutrophils. We also found that D-phenylalanine and D-tryptophan induced chemotactic responses in Jurkat cells transfected with the GPR109B cDNA but not in mock-transfected Jurkat cells. These results suggest that these aromatic D-amino acids elicit a chemotactic response in human neutrophils via activation of GPR109B. PMID:19237584

  6. Calcium-mediated increased expression of fibroblast growth factor-2 acts through NF-κB and PGE2/EP4 receptor signaling pathways in cementoblasts.

    PubMed

    Kanaya, Sousuke; Nemoto, Eiji; Sakisaka, Yukihiko; Shimauchi, Hidetoshi

    2013-10-01

    We reported previously that cementoblasts are provided with sensing mechanisms for extracellular Ca2+ and that elevated extracellular Ca2+ increases fibroblast growth factor-2 (FGF-2) gene and protein expression levels via a cyclic AMP/protein kinase A (PKA) dependent pathway. In the present study, we found that stimulation of murine cementoblasts with 10 mM CaCl2 induced cyclooxygenase-2 (COX-2) gene expression and prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced CaCl2-induced increase in Fgf-2 gene expression, indicating that PGE2 synthesized by COX-2 may be involved in FGF-2 induction. The inhibitory effect of NS-398 was restored completely by the addition of PGE2 receptor 4 (E-prostanoid receptor 4, called EP4) agonist, but not agonists for EP1, EP2, and EP3. Furthermore, EP4 antagonist significantly reduced CaCl2-induced Fgf-2 induction, suggesting that it is mediated by EP4 activation. However, stimulation with EP4 agonist alone in the absence of CaCl2 had no effect on the Fgf-2 induction, indicating that EP4 signaling alone is not sufficient. CaCl2 also upregulated gene expression levels of Ep4 and Cox-2, as well as Fgf-2 and induction of these genes was abolished by pretreatment with BMS-345541, a nuclear factor-κB (NF-κB) inhibitor, indicating that NF-κB signaling triggered by CaCl2 is indispensable for FGF-2 induction. Furthermore, CaCl2-induced Fgf-2 induction was synergistically enhanced by the addition of EP4 agonist. This indicates that the signaling triggered via CaCl2 and its combination with EP4 agonist may be useful as a novel strategy for periodontal regeneration. PMID:23851295

  7. The Secreted Form of Respiratory Syncytial Virus G Glycoprotein Helps the Virus Evade Antibody-Mediated Restriction of Replication by Acting as an Antigen Decoy and through Effects on Fc Receptor-Bearing Leukocytes▿

    PubMed Central

    Bukreyev, Alexander; Yang, Lijuan; Fricke, Jens; Cheng, Lily; Ward, Jerrold M.; Murphy, Brian R.; Collins, Peter L.

    2008-01-01

    Respiratory syncytial virus (RSV) readily infects and reinfects during infancy and throughout life, despite maternal antibodies and immunity from prior infection and without the need for significant antigenic change. RSV has two neutralization antigens, the F and G virion glycoproteins. G is expressed in both membrane-bound (mG) and secreted (sG) forms. We investigated whether sG might act as a decoy for neutralizing antibodies by comparing the in vitro neutralization of wild-type (wt) RSV versus recombinant mG RSV expressing only mG. wt RSV indeed was less susceptible than mG RSV to monovalent G-specific and polyvalent RSV-specific antibodies, whereas susceptibility to F-specific antibodies was equivalent. This difference disappeared when the virus preparations were purified to remove sG. Thus, sG appears to function as a neutralization decoy. We evaluated this effect in vivo in mice by comparing the effects of passively transferred antibodies on the pulmonary replication of wt RSV versus mG RSV. Again, wt RSV was less sensitive than mG RSV to G-specific and RSV-specific antibodies; however, a similar difference was also observed with F-specific antibodies. This confirmed that sG helps wt RSV evade the antibody-dependent restriction of replication but indicated that in mice, it is not acting primarily as a decoy for G-specific antibodies, perhaps because sG is produced in insufficient quantities in this poorly permissive animal. Rather, we found that the greater sensitivity of mG versus wt RSV to the antiviral effect of passively transferred RSV antibodies required the presence of inflammatory cells in the lung and was Fcγ receptor dependent. Thus, sG helps RSV escape the antibody-dependent restriction of replication via effects as an antigen decoy and as a modulator of leukocytes bearing Fcγ receptors. PMID:18842713

  8. Interleukin-1 Receptor Type 2 Acts with c-Fos to Enhance the Expression of Interleukin-6 and Vascular Endothelial Growth Factor A in Colon Cancer Cells and Induce Angiogenesis.

    PubMed

    Mar, Ai-Chung; Chu, Chun-Ho; Lee, Hui-Ju; Chien, Chia-Wen; Cheng, Jing-Jy; Yang, Shung-Haur; Jiang, Jeng-Kai; Lee, Te-Chang

    2015-09-01

    Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor of exogenous IL-1; however, its intracellular activity is poorly understood. We previously demonstrated that IL1R2 intracellularly activates the expression of several proinflammatory cytokines and affects cell migration. In this study, we found that intracellular IL1R2 expression was increased in human colorectal cancer cells (CRCs) compared with normal colon cells. We also observed that the mRNA levels of IL1R2 were highly correlated with IL-6 in tumor tissues of CRC patients. By modulating its expression in CRC cells, we verified that enhanced IL1R2 expression transcriptionally activated the expression of IL-6 and VEGF-A. Conditioned medium harvested from IL1R2-overexpressing CRC cells contained higher levels of IL-6 and VEGF-A than that from vector control cells and significantly enhanced the proliferation, migration, and tube formation of cultured endothelial cells. We further demonstrated a positive association of intracellular IL1R2 levels with tumor growth and microvessel density in xenograft mouse models. These results revealed that IL1R2 activates the expression of angiogenic factors. Mechanistically, we revealed that IL1R2 complexes with c-Fos and binds to the AP-1 site at the IL-6 and VEGF-A promoters. Together, these results reveal a novel function of intracellular IL1R2 that acts with c-Fos to enhance the transcription of IL-6 and VEGF-A, which promotes angiogenesis in CRC. PMID:26209639

  9. Sex Differences in Kappa Opioid Receptor Function and Their Potential Impact on Addiction

    PubMed Central

    Chartoff, Elena H.; Mavrikaki, Maria

    2015-01-01

    Behavioral, biological, and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN), an endogenous ligand at kappa opioid receptors (KORs), is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain, mood, and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN) gene, genetic linkage with the melanocortin-1 receptor (MC1R), heterodimerization of KORs and mu opioid receptors (MORs), and gonadal hormones. Finally, we

  10. Effects of mu- and kappa-2 opioid receptor agonists on pain and rearing behaviors

    PubMed Central

    Neubert, John K; Rossi, Heather L; Pogar, Jonathan; Jenkins, Alan C; Caudle, Robert M

    2007-01-01

    Background Management of pain involves a balance between inhibition of pain and minimization of side effects; therefore, in developing new analgesic compounds, one must consider the effects of treatment on both pain processing and behavior. The purpose of this study was to evaluate the effects of the mu and kappa-2 opioid receptor agonists on general and pain behavioral outcomes. Methods As a general behavioral assessment, we modified the cylinder rearing assay and recorded the number and duration of rearing events. Thermal sensitivity was evaluated using either a reflexive measure of hindpaw withdrawal latency to a radiant heat source or using an orofacial operant thermal assay. Acetic acid-induced visceral pain and capsaicin-induced neurogenic inflammatory pain were used as painful stimuli. The mu-opioid receptor agonist, morphine or the kappa-2 receptor agonist GR89696 was administered 30 min prior to testing. A general linear model repeated measures analysis was completed for baseline session comparisons and an analysis of variance was used to evaluate the effects of treatment on each outcome measure (SPSS Inc). When significant differences were found, post-hoc comparisons were made using the Tukey honestly significant difference test. *P < 0.05 was considered significant in all instances. Results We found that morphine and GR89,696 dose-dependently decreased the number of reaching events and rearing duration. Rearing behavior was not affected at 0.5 mg/kg for morphine, 1.25 × 10-4 mg/kg for GR89,696. Hindpaw thermal sensitivity was significantly increased only at the highest doses for each drug. At the highest dose that did not significantly influence rearing behavior, we found that visceral and neurogenic inflammatory pain was not affected following GR89,696 administration and morphine was only partially effective for blocking visceral pain. Conclusion This study demonstrated that high levels of the opioids produced significant untoward effects and made

  11. An Orphan G Protein-Coupled Receptor, GPR1, Acts as a Coreceptor To Allow Replication of Human Immunodeficiency Virus Types 1 and 2 in Brain-Derived Cells

    PubMed Central

    Shimizu, Nobuaki; Soda, Yasushi; Kanbe, Katsuaki; Liu, Hui-Yu; Jinno, Atsushi; Kitamura, Toshio; Hoshino, Hiroo

    1999-01-01

    Twelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1. These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains. However, a factor that determines this cell tropism has not been identified. This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2ROD strain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1. When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection. These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2ROD strain in vitro. PMID:10233994

  12. A Novel Glucagon-like Peptide-1 (GLP-1)/Glucagon Hybrid Peptide with Triple-acting Agonist Activity at Glucose-dependent Insulinotropic Polypeptide, GLP-1, and Glucagon Receptors and Therapeutic Potential in High Fat-fed Mice*

    PubMed Central

    Gault, Victor A.; Bhat, Vikas K.; Irwin, Nigel; Flatt, Peter R.

    2013-01-01

    Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA2]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA2]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA2]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA2]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA2]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA2]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA2]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes. PMID:24165127

  13. Novel hormone "receptors".

    PubMed

    Nemere, Ilka; Hintze, Korry

    2008-02-01

    Our concepts of hormone receptors have, until recently, been narrowly defined. In the last few years, an increasing number of reports identify novel proteins, such as enzymes, acting as receptors. In this review we cover the novel receptors for the hormones atrial naturetic hormone, enterostatin, hepcidin, thyroid hormones, estradiol, progesterone, and the vitamin D metabolites 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3). PMID:17546587

  14. Protease-activated receptor-1 (PAR1) acts via a novel Galpha13-dishevelled axis to stabilize beta-catenin levels.

    PubMed

    Turm, Hagit; Maoz, Myriam; Katz, Vered; Yin, Yong-Jun; Offermanns, Steffan; Bar-Shavit, Rachel

    2010-05-14

    We have previously shown a novel link between hPar-1 (human protease-activated receptor-1) and beta-catenin stabilization. Although it is well recognized that Wnt signaling leads to beta-catenin accumulation, the role of PAR1 in the process is unknown. We provide here evidence that PAR1 induces beta-catenin stabilization independent of Wnt, Fz (Frizzled), and the co-receptor LRP5/6 (low density lipoprotein-related protein 5/6) and identify selective mediators of the PAR1-beta-catenin axis. Immunohistological analyses of hPar1-transgenic (TG) mouse mammary tissues show the expression of both Galpha(12) and Galpha(13) compared with age-matched control counterparts. However, only Galpha(13) was found to be actively involved in PAR1-induced beta-catenin stabilization. Indeed, a dominant negative form of Galpha(13) inhibited both PAR1-induced Matrigel invasion and Lef/Tcf (lymphoid enhancer factor/T cell factor) transcription activity. PAR1-Galpha(13) association is followed by the recruitment of DVL (Dishevelled), an upstream Wnt signaling protein via the DIX domain. Small interfering RNA-Dvl silencing leads to a reduction in PAR1-induced Matrigel invasion, inhibition of Lef/Tcf transcription activity, and decreased beta-catenin accumulation. It is of note that PAR1 also promotes the binding of beta-arrestin-2 to DVL, suggesting a role for beta-arrestin-2 in PAR1-induced DVL phosphorylation dynamics. Although infection of small interfering RNA-LRP5/6 or the use of the Wnt antagonists, SFRP2 (soluble Frizzled-related protein 2) or SFRP5 potently reduced Wnt3A-mediated beta-catenin accumulation, no effect was observed on PAR1-induced beta-catenin stabilization. Collectively, our data show that PAR1 mediates beta-catenin stabilization independent of Wnt. We propose here a novel cascade of PAR1-induced Galpha(13)-DVL axis in cancer and beta-catenin stabilization. PMID:20223821

  15. Plasma membrane receptor-like kinase leaf panicle 2 acts downstream of the DROUGHT AND SALT TOLERANCE transcription factor to regulate drought sensitivity in rice

    PubMed Central

    Wu, Fuqing; Sheng, Peike; Tan, Junjie; Chen, Xiuling; Lu, Guangwen; Ma, Weiwei; Heng, Yueqin; Lin, Qibing; Zhu, Shanshan; Wang, Jiulin; Wang, Jie; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Wan, Jianmin

    2015-01-01

    Drought is a recurring climatic hazard that reduces the crop yields. To avoid the negative effects of drought on crop production, extensive efforts have been devoted to investigating the complex mechanisms of gene expression and signal transduction during drought stress. Receptor-like kinases (RLKs) play important roles in perceiving extracellular stimuli and activating downstream signalling responses. The rice genome contains >1100 RLK genes, of which only two are reported to function in drought stress. A leucine-rich repeat (LRR)-RLK gene named Leaf Panicle 2 (LP2) was previously found to be strongly expressed in leaves and other photosynthetic tissues, but its function remains unclear. In the present study, it was shown that the expression of LP2 was down-regulated by drought and abscisic acid (ABA). Transgenic plants overexpressing LP2 accumulated less H2O2, had more open stomata in leaves, and showed hypersensitivity to drought stress. Further investigation revealed that transcription of LP2 was directly regulated by the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST). In addition, LP2 was identified as a functional kinase localized to the plasma membrane and interacted with the drought-responsive aquaporin proteins OsPIP1; 1, OsPIP1; 3, and OsPIP2; 3. Thus, the findings provided evidence that the LRR-RLK LP2, transcriptionally regulated by the drought-related transcription factor DST, served as a negative regulator in drought response. PMID:25385766

  16. Systemic injection of CK2.3, a novel peptide acting downstream of Bone Morphogenetic Protein receptor BMPRIa, leads to increased trabecular bone mass

    PubMed Central

    Akkiraju, Hemanth; Bonor, Jeremy; Olli, Kristine; Bowen, Chris; Bragdon, Beth; Coombs, Harold; Donahue, Leah Rae; Duncan, Randall; Nohe, Anja

    2014-01-01

    Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal-related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation. PMID:25331517

  17. Nucleotide-binding oligomerization domain 1 acts in concert with the cholecystokinin receptor agonist, cerulein, to induce IL-33-dependent chronic pancreatitis.

    PubMed

    Watanabe, T; Sadakane, Y; Yagama, N; Sakurai, T; Ezoe, H; Kudo, M; Chiba, T; Strober, W

    2016-09-01

    Nucleotide-binding oligomerization domain 1 (NOD1) fulfills important host-defense functions via its responses to a variety of gut pathogens. Recently, however, we showed that in acute pancreatitis caused by administration of cholecystokinin receptor (CCKR) agonist (cerulein) NOD1 also has a role in inflammation via its responses to gut commensal organisms. In the present study, we explored the long-term outcome of such NOD1 responsiveness in a new model of chronic pancreatitis induced by repeated administration of low doses of cerulein in combination with NOD1 ligand. We found that the development of chronic pancreatitis in this model requires intact NOD1 and type I IFN signaling and that such signaling mediates a macrophage-mediated inflammatory response that supports interleukin (IL)-33 production by acinar cells. The IL-33, in turn, has a necessary role in the induction of IL-13 and TGF-β1, factors causing the fibrotic reaction characteristic of chronic pancreatitis. Interestingly, the Th2 effects of IL-33 were attenuated by the concomitant type I IFN response since the inflammation was marked by clear increases in IFN-γ and TNF-α production but only marginal increases in IL-4 production. These studies establish chronic pancreatitis as an IL-33-dependent inflammation resulting from synergistic interactions between the NOD1 and CCKR signaling pathways. PMID:26813347

  18. Macitentan (ACT-064992), a Tissue-Targeting Endothelin Receptor Antagonist, Enhances Therapeutic Efficacy of Paclitaxel by Modulating Survival Pathways in Orthotopic Models of Metastatic Human Ovarian Cancer12

    PubMed Central

    Kim, Sun-Jin; Kim, Jang Seong; Kim, Seung Wook; Brantley, Emily; Yun, Seok Joong; He, Junqin; Maya, Marva; Zhang, Fahao; Wu, Qiuyu; Lehembre, François; Regenass, Urs; Fidler, Isaiah J

    2011-01-01

    Potential treatments for ovarian cancers that have become resistant to standard chemotherapies include modulators of tumor cell survival, such as endothelin receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1, growing orthotopically in nude mice. Mice with established disease were treated with vehicle (control), paclitaxel (weekly, intraperitoneal injections), macitentan (daily oral administrations), or a combination of paclitaxel and macitentan. Treatment with paclitaxel decreased tumor weight and volume of ascites. Combination therapy with macitentan and paclitaxel reduced tumor incidence and further reduced tumor weight and volume of ascites when compared with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but alone had no effect on tumor incidence or ascites. Immunohistochemical analyses revealed that treatment with macitentan and macitentan plus paclitaxel inhibited the phosphorylation of ETRs and suppressed the survival pathways of tumor cells by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan necessary for inhibition of phosphorylation correlated with the dose required to increase antitumor efficacy of paclitaxel. Treatment with macitentan enhanced the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis in tumor cells and tumor-associated endothelial cells. Collectively, these results show that administration of macitentan in combination with paclitaxel prevents the progression of ovarian cancer in the peritoneal cavity of nude mice in part by inhibiting survival pathways of both tumor cells and tumor-associated endothelial cells. PMID:21403842

  19. The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy

    PubMed Central

    Secreto, Charla; Boysen, Justin; Sassoon, Traci; Shanafelt, Tait D.; Mukhopadhyay, Debabrata; Kay, Neil E.

    2011-01-01

    Recently, we detected that chronic lymphocytic leukemia (CLL) B-cell–derived microvesicles in CLL plasma carry a constitutively phosphorylated novel receptor tyrosine kinase (RTK), Axl, indicating that Axl was acquired from the leukemic B cells. To examine Axl status in CLL, we determined the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B cells by Western blot analysis. We detected differential levels of P-Axl in CLL B cells, and further analysis showed that expression of P-Axl was correlated with the other constitutively phosphorylated kinases, including Lyn, phosphoinositide-3 kinase, SyK/ζ-associated protein of 70 kDa, phospholipase C γ2 in CLL B cells. We found that these intracellular signaling molecules were complexed with P-Axl in primary CLL B cells. When Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, bosutinib (SKI-606), or a specific-inhibitor of Axl (R428), robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner. Therefore, we have identified a novel RTK in CLL B cells which appears to work as a docking site for multiple non-RTKs and drives leukemic cell survival signals. These findings highlight a unique target for CLL treatment. PMID:21135257

  20. Plasma membrane receptor-like kinase leaf panicle 2 acts downstream of the DROUGHT AND SALT TOLERANCE transcription factor to regulate drought sensitivity in rice.

    PubMed

    Wu, Fuqing; Sheng, Peike; Tan, Junjie; Chen, Xiuling; Lu, Guangwen; Ma, Weiwei; Heng, Yueqin; Lin, Qibing; Zhu, Shanshan; Wang, Jiulin; Wang, Jie; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Wan, Jianmin

    2015-01-01

    Drought is a recurring climatic hazard that reduces the crop yields. To avoid the negative effects of drought on crop production, extensive efforts have been devoted to investigating the complex mechanisms of gene expression and signal transduction during drought stress. Receptor-like kinases (RLKs) play important roles in perceiving extracellular stimuli and activating downstream signalling responses. The rice genome contains >1100 RLK genes, of which only two are reported to function in drought stress. A leucine-rich repeat (LRR)-RLK gene named Leaf Panicle 2 (LP2) was previously found to be strongly expressed in leaves and other photosynthetic tissues, but its function remains unclear. In the present study, it was shown that the expression of LP2 was down-regulated by drought and abscisic acid (ABA). Transgenic plants overexpressing LP2 accumulated less H₂O₂, had more open stomata in leaves, and showed hypersensitivity to drought stress. Further investigation revealed that transcription of LP2 was directly regulated by the zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST). In addition, LP2 was identified as a functional kinase localized to the plasma membrane and interacted with the drought-responsive aquaporin proteins OsPIP1; 1, OsPIP1; 3, and OsPIP2; 3. Thus, the findings provided evidence that the LRR-RLK LP2, transcriptionally regulated by the drought-related transcription factor DST, served as a negative regulator in drought response. PMID:25385766

  1. Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure

    PubMed Central

    Zhang, Li; Ren, Feng; Zhang, Xiangying; Wang, Xinxin; Shi, Hongbo; Zhou, Li; Zheng, Sujun; Chen, Yu; Chen, Dexi; Li, Liying; Duan, Zhongping

    2016-01-01

    ABSTRACT Peroxisome proliferator-activated receptor α (PPARα) is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF). However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress) plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN)- and lipopolysaccharide (LPS)-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA) was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1) PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78), Grp94 and C/EBP-homologous protein (CHOP) in vivo; (2) the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA) treatment reversed liver protection and increased hepatocyte apoptosis; (3) in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF. PMID:27482818

  2. The Nuclear Hormone Receptor NHR-40 Acts Downstream of the Sulfatase EUD-1 as Part of a Developmental Plasticity Switch in Pristionchus.

    PubMed

    Kieninger, Manuela R; Ivers, Nicholas A; Rödelsperger, Christian; Markov, Gabriel V; Sommer, Ralf J; Ragsdale, Erik J

    2016-08-22

    Developmental plasticity, the ability of one genotype to produce distinct phenotypes in different environments, has been suggested to facilitate phenotypic diversification, and several examples in plants and animals support its macroevolutionary potential [1-8]. However, little is known about associated molecular mechanisms, because environmental effects on development are difficult to study by laboratory approaches. One promising system is the mouth dimorphism of the nematode Pristionchus pacificus [9-12]. Following an irreversible decision in larval development, these nematodes form moveable teeth that occur in either of two discrete morphs. The "eurystomatous" (Eu) form has a wide mouth and two teeth, allowing predatory feeding on other nematodes. In contrast, the alternative ("stenostomatous"; St) form has diminutive mouthparts that largely constrain its diet to microbes. The sulfatase EUD-1 was previously discovered to execute a polyphenism switch based on dosage of functional alleles [13] and confirmed a prediction of evolutionary theory about how developmental switches control plasticity [1, 3]. However, the genetic context of this single gene, and hence the molecular complexity of switch mechanisms, was previously unknown. Here we use a suppressor screen to identify factors downstream of eud-1 in mouth-form regulation. We isolated three dominant, X-linked mutants in the nuclear hormone receptor gene nhr-40 that are haploinsufficient. Both eud-1 nhr-40 double and nhr-40 single mutants are all Eu, whereas transgenic overexpression of nhr-40 does not restore the wild-type phenotype but instead results in nearly all-St lines. Thus, NHR-40 is part of a developmental switch, suggesting that switch mechanisms controlling plasticity consist of multi-component hormonal signaling systems. PMID:27451902

  3. Marine Natural Products Acting on the Acetylcholine-Binding Protein and Nicotinic Receptors: From Computer Modeling to Binding Studies and Electrophysiology

    PubMed Central

    Kudryavtsev, Denis; Makarieva, Tatyana; Utkina, Natalia; Santalova, Elena; Kryukova, Elena; Methfessel, Christoph; Tsetlin, Victor; Stonik, Valentin; Kasheverov, Igor

    2014-01-01

    For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([125I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (Ki 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH4C1 cell line, no competition with [125I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (Ki ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources. PMID:24686559

  4. Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure.

    PubMed

    Zhang, Li; Ren, Feng; Zhang, Xiangying; Wang, Xinxin; Shi, Hongbo; Zhou, Li; Zheng, Sujun; Chen, Yu; Chen, Dexi; Li, Liying; Zhao, Caiyan; Duan, Zhongping

    2016-07-01

    Peroxisome proliferator-activated receptor α (PPARα) is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF). However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress) plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN)- and lipopolysaccharide (LPS)-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA) was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1) PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78), Grp94 and C/EBP-homologous protein (CHOP) in vivo; (2) the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA) treatment reversed liver protection and increased hepatocyte apoptosis; (3) in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF. PMID:27482818

  5. {beta}-Catenin can act as a nuclear import receptor for its partner transcription factor, lymphocyte enhancer factor-1 (lef-1)

    SciTech Connect

    Asally, Munehiro; Yoneda, Yoshihiro . E-mail: yyoneda@anat3.med.osaka-u.ac.jp

    2005-08-15

    Nuclear accumulation of {beta}-catenin plays an important role in the Wnt signaling pathway. In the nucleus, {beta}-catenin acts as a transcriptional co-activator for TCF/LEF family of transcription factors. It has been shown that lef-1 contains a typical basic type nuclear localization signal (NLS) and is transported into the nucleus by the conventional import pathway. In this study, we found that a mutant lef-1 lacking the classical NLS accumulated in the nucleus of living cells, when {beta}-catenin was co-expressed. In addition, in a cell-free import assay, lef-1 migrated into the nucleus in the presence of {beta}-catenin alone without any other soluble factors. In contrast, another mutant lef-1 lacking the {beta}-catenin binding domain failed to migrate into the nucleus, even in the presence of {beta}-catenin. These findings indicate that {beta}-catenin alone can mediate the nuclear import of lef-1 through the direct binding. Collectively, we propose that there are two distinct pathways for the nuclear import of lef-1: importin {alpha}/{beta}-mediated and {beta}-catenin-mediated one, which provides a novel paradigm for Wnt signaling pathway.

  6. Determination of landiolol, an ultra-short-acting β₁-receptor antagonist, in human plasma by liquid chromatography-tandem mass spectrometry.

    PubMed

    He, Qun; Shi, Meiyun; Liu, Xidong; Sun, Yantong; Hu, Lianghai; Yang, Yan; Fawcett, J Paul; Gu, Jingkai; Zhao, Limei

    2012-04-01

    A method for the determination of landiolol, an ultra-short-acting β₁-adrenoreceptor antagonist, in human plasma has been developed and validated. With the addition of pyridostigmine bromide to stabilize landiolol in the blood/plasma samples, and bisoprolol as internal standard, plasma samples were subjected to liquid-liquid extraction with diethyl ether:dicholoromethane (60:40, v/v) prior to assay by liquid chromatography-tandem mass spectrometry. Separation was performed on a TC-C₁₈ column (150 mm × 4.6 mm, 5 μm) using a mobile phase of methanol:10 mM ammonium acetate containing 1% formic acid (65:35, v/v) in a run time of 3.5 min. Detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the precursor-to-product ion transitions of landiolol at m/z 510.1→157.2 and bisoprolol at m/z 326.3→116.1. The method was linear over the concentration range 0.5-500 ng/ml with a lower limit of quantitation of 0.5 ng/ml. Intra- and inter-day precisions (as relative standard deviation, RSD) were <4.4% and <10.0%, respectively, with accuracy (as relative error, RE) <10.0%. The method was successfully applied to a clinical pharmacokinetic study involving a continuous infusion of landiolol hydrochloride to healthy Chinese volunteers. PMID:22418070

  7. The macrocyclic tetrapeptide [D-Trp]CJ-15,208 produces short-acting κ opioid receptor antagonism in the CNS after oral administration

    PubMed Central

    Eans, Shainnel O; Ganno, Michelle L; Reilley, Kate J; Patkar, Kshitij A; Senadheera, Sanjeewa N; Aldrich, Jane V; McLaughlin, Jay P

    2013-01-01

    Background and Purpose Cyclic peptides are resistant to proteolytic cleavage, therefore potentially exhibiting activity after systemic administration. We hypothesized that the macrocyclic κ opioid receptor (KOR)-selective antagonist [D-Trp]CJ-15,208 would demonstrate antagonist activity after systemic, that is, s.c. and oral (per os, p. o.), administration. Experimental Approach C57BL/6J mice were pretreated with [D-Trp]CJ-15,208 s.c. or p.o. before administration of the KOR-selective agonist U50,488 and the determination of antinociception in the warm-water tail-withdrawal assay. The locomotor activity of mice treated with [D-Trp]CJ-15,208 was determined by rotorod testing. Additional mice demonstrating cocaine conditioned place preference and subsequent extinction were pretreated daily with vehicle or [D-Trp]CJ-15,208 and then exposed to repeated forced swim stress or a single additional session of cocaine place conditioning before redetermining place preference. Key Results Pretreatment with [D-Trp]CJ-15,208 administered s.c. or p.o. dose-dependently antagonized the antinociception induced by i.p. administration of U50,488 in mice tested in the warm-water tail-withdrawal assay for less than 12 and 6 h respectively. [D-Trp]CJ-15,208 also produced limited (<25%), short-duration antinociception mediated through KOR agonism. Orally administered [D-Trp]CJ-15,208 dose-dependently antagonized centrally administered U50,488-induced antinociception, and prevented stress-, but not cocaine-induced, reinstatement of extinguished cocaine-seeking behaviour, consistent with its KOR antagonist activity, without affecting locomotor activity. Conclusions and Implications The macrocyclic tetrapeptide [D-Trp]CJ-15,208 is a short-duration KOR antagonist with weak KOR agonist activity that is active after oral administration and demonstrates blood–brain barrier permeability. These data validate the use of systemically active peptides such as [D-Trp]CJ-15,208 as potentially useful

  8. Pharmacological characterization of GSK573719 (umeclidinium): a novel, long-acting, inhaled antagonist of the muscarinic cholinergic receptors for treatment of pulmonary diseases.

    PubMed

    Salmon, Michael; Luttmann, Mark A; Foley, James J; Buckley, Peter T; Schmidt, Dulcie B; Burman, Miriam; Webb, Edward F; DeHaas, Christopher J; Kotzer, Charles J; Barrett, Victoria J; Slack, Robert J; Sarau, Henry M; Palovich, Michael R; Lainé, Dramane I; Hay, Douglas W P; Rumsey, William L

    2013-05-01

    Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 μg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 μg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases. PMID:23435542

  9. Hormonal regulation of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus

    PubMed Central

    Williams, Tanya J.; Torres-Reveron, Annelyn; Chapleau, Jeanette D.; Milner, Teresa A.

    2011-01-01

    Clinical and preclinical studies indicate that women and men differ in relapse vulnerability to drug-seeking behavior during abstinence periods. As relapse is frequently triggered by exposure of the recovered addict to objects previously associated with drug use and the formation of these associations requires memory systems engaged by the hippocampal formation (HF), studies exploring ovarian hormone modulation of hippocampal function are warranted. Previous studies revealed that ovarian steroids alter endogenous opioid peptide levels and trafficking of mu opioid receptors in the HF, suggesting cooperative interaction between opioids and estrogens in modulating hippocampal excitability. However, whether ovarian steroids affect the levels or trafficking of delta opioid receptors (DORs) in the HF is unknown. Here, hippocampal sections of adult male and normal cycling female Sprague-Dawley rats were processed for quantitative immunoperoxidase light microscopy and dual label fluorescence or immunoelectron microscopy using antisera directed against the DOR and neuropeptide Y (NPY). Consistent with previous studies in males, DOR-immunoreactivity (-ir) localized to select interneurons and principal cells in the female HF. In comparison to males, females, regardless of estrous cycle phase, show reduced DOR-ir in the granule cell layer of the dentate gyrus and proestrus (high estrogen) females, in particular, display reduced DOR-ir in the CA1 pyramidal cell layer. Ultrastructural analysis of DOR-labeled profiles in CA1 revealed that while females generally show fewer DORs in the distal apical dendrites of pyramidal cells, proestrus females, in particular, exhibit DOR internalization and trafficking towards the soma. Dual label studies revealed that DORs are found in NPY-labeled interneurons in the hilus, CA3, and CA1. While DOR colocalization frequency in NPY-labeled neuron somata was similar between animals in the hilus, proestrus females had fewer NPY-labeled neurons that

  10. The disease-modifying effects of a Sativex-like combination of phytocannabinoids in mice with experimental autoimmune encephalomyelitis are preferentially due to Δ9-tetrahydrocannabinol acting through CB1 receptors.

    PubMed

    Moreno-Martet, Miguel; Feliú, Ana; Espejo-Porras, Francisco; Mecha, Miriam; Carrillo-Salinas, Francisco J; Fernández-Ruiz, Javier; Guaza, Carmen; de Lago, Eva

    2015-11-01

    Sativex(®), an equimolecular combination of Δ(9)-tetrahydrocannabinol-botanical drug substance (Δ(9)-THC-BDS) and cannabidiol-botanical drug substance (CBD-BDS), is a licensed medicine that may be prescribed for alleviating specific symptoms of multiple sclerosis (MS) such as spasticity and pain. However, further evidence suggest that it could be also active as disease-modifying therapy given the immunomodulatory, anti-inflammatory and cytoprotective properties of their two major components. In this study, we investigated this potential in the experimental autoimmune encephalitis (EAE) model of MS in mice. We compared the effect of a Sativex-like combination of Δ(9)-THC-BDS (10 mg/kg) and CBD-BDS (10 mg/kg) with Δ(9)-THC-BDS (20 mg/kg) or CBD-BDS (20 mg/kg) administered separately by intraperitoneal administration to EAE mice. Treatments were initiated at the time that symptoms appear and continued up to the first relapse of the disease. The results show that the treatment with a Sativex-like combination significantly improved the neurological deficits typical of EAE mice, in parallel with a reduction in the number and extent of cell aggregates present in the spinal cord which derived from cell infiltration to the CNS. These effects were completely reproduced by the treatment with Δ(9)-THC-BDS alone, but not by CBD-BDS alone which only delayed the onset of the disease without improving disease progression and reducing the cell infiltrates in the spinal cord. Next, we investigated the potential targets involved in the effects of Δ(9)-THC-BDS by selectively blocking CB(1) or PPAR-γ receptors, and we found a complete reversion of neurological benefits and the reduction in cell aggregates only with rimonabant, a selective CB(1) receptor antagonist. Collectively, our data support the therapeutic potential of Sativex as a phytocannabinoid formulation capable of attenuating EAE progression, and that the active compound was Δ(9)-THC-BDS acting through CB(1

  11. DEVELOPMENT AND IN VITRO CHARACTERIZATION OF A NOVEL BIFUNCTIONAL MU-AGONIST/DELTA-ANTAGONIST OPIOID TETRAPEPTIDE

    PubMed Central

    Purington, Lauren C.; Sobczyk-Kojiro, Katarzyna; Pogozheva, Irina D.; Traynor, John R.; Mosberg, Henry I.

    2011-01-01

    The development of tolerance to and dependence on opioid analgesics greatly reduces their long-term usefulness. Previous studies have demonstrated that co-administration of a mu opioid receptor (MOR) agonist and delta opioid receptor (DOR) antagonist can decrease MOR agonist induced tolerance and dependence development after chronic exposure. Clinically, a single ligand displaying multiple efficacies (e.g. MOR agonism concurrently with DOR antagonism) would be of increased value over two drugs administered simultaneously. Guided by modeling of receptor-ligand complexes we have developed a series of potent non-selective opioid tetrapeptides that have differing efficacy at MOR and DOR. In particular, our lead peptide (KSK-103) binds with equal affinity to MOR and DOR but acts as a MOR agonist with similar efficacy but greater potency than morphine and a DOR antagonist in cellular assays measuring both G protein stimulation and adenylyl cyclase inhibition. PMID:21958158

  12. Clinical utility of naloxegol in the treatment of opioid-induced constipation

    PubMed Central

    Bruner, Heather C; Atayee, Rabia S; Edmonds, Kyle P; Buckholz, Gary T

    2015-01-01

    Opioids are a class of medications frequently used for the treatment of acute and chronic pain, exerting their desired effects at central opioid receptors. Agonism at peripherally located opioid receptors, however, leads to opioid-induced constipation (OIC), one of the most frequent and debilitating side effects of prolonged opioid use. Insufficient relief of OIC with lifestyle modification and traditional laxative treatments may lead to decreased compliance with opioid regimens and undertreated pain. Peripherally acting mu-opioid receptor antagonists (PAMORAs) offer the reversal of OIC without loss of central pain relief. Until recently, PAMORAs were restricted to subcutaneous route or to narrow patient populations. Naloxegol is the first orally dosed PAMORA indicated for the treatment of OIC in noncancer patients. Studies have suggested its efficacy in patients failing traditional constipation treatments; however, insufficient evidence exists to establish its role in primary prevention of OIC at this time. PMID:26109876