DOCSLIB.ORG

A,-, and P-Opioid Receptor Agonists on Excitatory Transmission in Lamina II Neurons of Adult Rat Spinal Cord

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A,-, and P-Opioid Receptor Agonists on Excitatory Transmission in Lamina II Neurons of Adult Rat Spinal Cord The Journal of Neuroscience, August 1994, 74(E): 4965-4971 Inhibitory Actions of S,-, a,-, and p-Opioid Receptor Agonists on Excitatory Transmission in Lamina II Neurons of Adult Rat Spinal Cord Steven R. Glaum,’ Richard J. Miller,’ and Donna L. Hammond* Departments of lPharmacoloaical and Phvsioloqical Sciences and ‘Anesthesia and Critical Care, The University of Chicago, Chicago, Illinois 60637 . This study examined the electrophysiological consequences tor in rat spinal cord and indicate that activation of either of selective activation of 6,-, 6,-, or r-opioid receptors using 6,- or Qopioid receptors inhibits excitatory, glutamatergic whole-cell recordings made from visually identified lamina afferent transmission in the spinal cord. This effect may me- II neurons in thin transverse slices of young adult rat lumbar diate the ability of 6, or 6, receptor agonists to produce an- spinal cord. Excitatory postsynaptic currents (EPSCs) or po- tinociception when administered intrathecally in the rat. tentials (EPSPs) were evoked electrically at the ipsilateral [Key words: DPDPE, deltorphin, spinal cord slice, EPSP, dorsal root entry zone after blocking inhibitory inputs with 6-opioid receptor, DAMGO, naltriben, 7-benzylidene- bicuculline and strychnine, and NMDA receptors with o-2- naltrexone (BNTX), naloxone] amino+phosphonopentanoic acid. Bath application of the p receptor agonist [D-Ala2, KMePhe4, Gly5-ollenkephalin (DAMGO) or the 6, receptor agonist [D-Pen2, o-PerF]en- The dorsal horn of the spinal cord is an important site for the kephalin (DPDPE) produced a log-linear, concentration-de- production of antinociception by K- and 6-opioid receptor ag- pendent reduction in the amplitude of the evoked EPSP/ onists (Yaksh, 1993). Postsynaptic, as well as presynaptic, sites EPSC. By comparison, the 6, receptor agonist [D-Ala2, of action are thought to mediate this effect. A postsynaptic site Glu4]deltorphin (DELT) was unable to reduce the evoked of action is suggestedby the existence of enkephalinergic syn- EPSP/EPSC by more than 50% at 100 PM, the highest con- apseson dorsal horn neurons (Hunt et al., 1980; Aronin et al., centration tested. At concentrations that reduced evoked 1981; Ruda, 1982; Sumal et al., 1982) and by the ability of EPSP/EPSCs by 40-60%, neither DAMGO, DPDPE, nor DELT opioid receptor agoniststo inhibit the excitation of dorsal horn decreased the amplitude of the postsynaptic current pro- neurons produced by iontophoretic application of excitatory duced by brief pressure ejection of (S)-cu-amino-3-hydroxy- amino acids (Belcher and Ryall, 1978; Zieglgansbergerand Tul- 5-methyl-4-isoxazole-propionic acid, suggesting a presyn- loch, 1979; Willcockson et al., 1984). In addition, w-opioid re- aptic site of action of these opioid receptor agonists. Bath ceptor agonists hyperpolarize and decreasethe input resistance application of 200 nh! naltriben (NTB), a 8, receptor antago- of dorsal horn neurons in slicesof the rat spinal cord or spinal nist, competitively increased the EC,, of DELT by 15.3-fold, trigeminal nucleus (Murase et al., 1982; Jeftinija, 1988; Grudt but did not antagonize either DPDPE or DAMGO. The EC,, and Williams, 1994) primarily by increasing one or more K+ of DELT was further increased by 169.7-fold in the presence conductances (Yoshimura and North, 1983; Grudt and Wil- of 1 PM NTB. However, this high concentration of NTB also liams, 1994). A presynaptic site of action is suggestedby the increased the EC,, of DPDPE by about threefold in a non- ability of opioid receptor agoniststo inhibit the releaseof neu- competitive manner and antagonized DAMGO in a noncom- rotransmitters from primary afferent neurons (Go and Yaksh, petitive manner. In contrast, bath application of 33 or 100 1987; Collin et al., 199 1; Kangrga and RandiC, 1991) and by nM 7-benzylidenenaltrexone (BNTX), a 6, receptor antago- the marked decreasein the number of opioid receptor binding nist, produced a concentration-dependent, noncompetitive sites in the dorsal horn that occurs after dorsal rhizotomy or antagonism of DPDPE, but did not antagonize DELT. A mod- the destruction of small diameter primary afferents by capsaicin est noncompetitive antagonism of DAMGO occurred in the (reviewed by Bessonet al., 1989). In addition, low Ca’+/high presence of 100 nM BNTX. Bath application of 500 nM nal- Mg’+-containing solutions reduce the inhibitory effectsof opioid oxone competitively antagonized DAMGO as well as DPDPE, receptor agonists on synaptic transmission in the dorsal horn increasing their EC,, values by 13.3- and 2.5-fold, respec- (Murase et al., 1982; Hori et al., 1992). Finally, opioid receptor tively. These results provide the first electrophysiological agonists decreaseboth the frequency of miniature EPSPs and demonstration of functional subtypes of the &opioid recep- the amplitude ofevoked EPSPsin dorsal horn neurons in slices of rat spinal cord (Hori et al., 1992). Received July 7, 1993; revised Jan. 14, 1994; accepted Feb. 17, 1994. These data provide strong support for opioid modulation of This work was supported by United States Public Health Service Grants DA excitatory afferent synaptic transmissionin the spinal cord, par- 06736 to D.L.H. and DA 02575 and DA 02121 to R.J.M. ticularly by N-opioid receptors. However, the role of &opioid Correspondence should be addressed to Dr. Steven R. Glaum, Department of Pharmacological and Physiological Sciences, The University of Chicago, 947 East receptors is lessclear. The prototypic d-opioid receptor agonist 58th Street, Chicago IL 60637. [D-Pet?, o-Pen5]enkephalin (DPDPE) does not affect the mem- Copyright 0 1994 Society for Neuroscience 0270-6474/94/144965-07$05.00/O brane potential or the amplitude of evoked EPSPsrecorded in 4966 Glaum et al. Inhibitory Actions of 6- and a-Opioid Agonists modulation of excitatory afferent synaptic transmission in the control spinal cord. Whole-cell recordings were made of visually iden- tified neurons in lamina II, a principal termination site for small diameter primary afferent fibers that convey nociceptive infor- mation to the central nervous system (reviewed by Willis and Coggeshall, 1991; Light, 1992) in thin transverse slicesof the spinal cord obtained from young adult rats. The ability of the 6, receptor agonist DPDPE (Jiang et al., 1991; Sofuoglu et al., 1991, 1993) the 6, receptor agonist [D-Ala’, Glu4]deltorphin (DELT) (Jiang et al., 1991; Sofuoglu et al., 1991; Stewart and Hammond, 1993) or the p receptor agonist [D-Ala’, N-MePhe4, Glys-ollenkephalin (DAMGO) to inhibit evoked excitatory postsynaptic potentials/currents (EPSP/EPSCs)in theseneurons DAMGO was determined. In addition, the pharmacologic specificity of this inhibition wascharacterized using the 6, receptor antagonist 7-benzylidenenaltrexone (BNTX) (Portogheseet al., 1992; So- fuoglu et al., 1993) and the 6, receptor antagonist naltriben (NTB) (Sofuoglu et al., 199 1; Stewart and Hammond, 1993). -u Materials and Methods Slice preparation. Transverse slices of the rat spinal cord were prepared as previously described (Bleakman et al., 1992). Briefly, Sprague-Daw- recovery ley rats (Holtzman, Madison, WI) of either sex, aged between 28 and 60 d, were deeply anesthetized with ether and a laminectomy was per- formed that exposed the entire thoracolumbar enlargement and an ad- ditional three to four segments rostrocaudally. The rat was then killed “1 /y”“““” by ether overdose, and the spinal cord and surrounding tissue were immediately removed to a dissecting dish. The spinal cord was freed from the surrounding tissue and 175-225 Frn transverse slices were prepared from the region of the thoracolumbar enlargement with a vibrating microtome. Slices were allowed to recover for at least 1 hr in 32°C artificial cerebrospinal fluid (ACSF), which contained (in mM) NaCl, 126; NaHCO,, 26.2; NaH?PO,, 1; KCI, 3; MgSO,, 1.5; CaC&, 2.5; glucose, 10, and was continuously gassed with 95% OZ, 5% COZ. A single slice was then transferred to the recording chamber (volume of 1 ml), where Figure I. High-resolution trace of monosynaptic EPSCs evoked in a it was continuously perfused (4-6 ml min ‘) at room temperature with lamina II neuron and its inhibition by 0.3 PM DAMGO. The neuron ACSF containing 50 +M o-2-amino-5-phosphonopentanoic acid, 10 PM was voltage clamped at -70 mV and EPSCs were evoked at 0.1 Hz. bicuculline, and 10 PM strychnine. Under these conditions, a complete The averaged (n = 5) trace of the EPSC is shown immediately before exchange ofperfusate occurred in IO-25 sec. Whole-cell recordings were application of DAMGO in the perfusate (control), I5 set after DAMGO made with 2-5 Mq patch electrodes containing in mM: K-gluconate, application (DAMGO), and following a 2 min washout (recovery). The 145; MgCI,, 2; CaCI,, 0.1; HEPES, 5; EGTA, 1.1; K,ATP, 5 (pH 7.2). stimulus artifact has been retouched in the figure. Experiments were typically performed under voltage-clamp conditions in the discontinuous mode of the amplifier at a holding potential of -60 to -80 mV. In discontinuous current-clamp experiments, resting k’,,, was held constant at -60 to -80 mV by direct current injection. superficial dorsal horn neurons in slices of rat spinal cord or Monosynaptic EPSP/EPSCs were evoked by single, subthreshold stimuli spinal trigeminal nucleus (Jeftinija, 1988; Grudt and Williams, (30-120 psec, 3-10 V) delivered at 0.1-0.2 Hz by a bipolar tungsten 1994). Similarly, [D-Pen’, L-Pen5]enkephalin,a close structural electrode placed in the ipsilateral dorsal root entry zone. These EPSP/ analog of DPDPE, does not affect the frequency of miniature EPSCs are denoted generically as EPSCs in the subsequent text. Addition of 10 PM 6,7-dinitroquinoxaline-2,3-dione completely suppressed the EPSPsrecorded in lamina I neurons in slicesof rat spinal cord evoked EPSCs, indicating they were mediated by the actions of gluta- (Hori et al., 1992) leading the authors to conclude that excit- mate on the ol-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid atory synaptic transmission is predominantly modulated by fi (AMPA)/kainate receptor subtype (Schneider and Perl, 1988; Yoshi- receptors(Jeftinija, 1988; Hori et al., 1992).
Load more

Recommended publications
  • (12) Patent Application Publication (10) Pub. No.: US 2004/0224020 A1 Schoenhard (43) Pub
    US 2004O224020A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0224020 A1 Schoenhard (43) Pub. Date: Nov. 11, 2004 (54) ORAL DOSAGE FORMS WITH (22) Filed: Dec. 18, 2003 THERAPEUTICALLY ACTIVE AGENTS IN CONTROLLED RELEASE CORES AND Related U.S. Application Data IMMEDIATE RELEASE GELATIN CAPSULE COATS (60) Provisional application No. 60/434,839, filed on Dec. 18, 2002. (76) Inventor: Grant L. Schoenhard, San Carlos, CA (US) Publication Classification Correspondence Address: (51) Int. Cl. ................................................... A61K 9/24 Janet M. McNicholas (52) U.S. Cl. .............................................................. 424/471 McAndrews, Held & Malloy, Ltd. 34th Floor (57) ABSTRACT 500 W. Madison Street Chicago, IL 60661 (US) The present invention relates to oral dosage form with active agents in controlled release cores and in immediate release (21) Appl. No.: 10/742,672 gelatin capsule coats. Patent Application Publication Nov. 11, 2004 Sheet 1 of 3 US 2004/0224020 A1 r N 2.S Hr s Patent Application Publication Nov. 11, 2004 Sheet 2 of 3 US 2004/0224020 A1 r CN -8 e N va N . t Cd NOLLYRILNONOO Patent Application Publication Nov. 11, 2004 Sheet 3 of 3 US 2004/0224020 A1 US 2004/0224020 A1 Nov. 11, 2004 ORAL DOSAGE FORMS WITH released formulations, a long t is particularly disadvan THERAPEUTICALLY ACTIVE AGENTS IN tageous to patients Seeking urgent treatment and to maintain CONTROLLED RELEASE CORES AND MEC levels. A second difference in the pharmacokinetic IMMEDIATE RELEASE GELATIN CAPSULE profiles of controlled release in comparison to immediate COATS release drug formulations is that the duration of Sustained plasma levels is longer in the controlled release formula CROSS REFERENCED APPLICATIONS tions.
    [Show full text]
  • TABLE 1 Studies of Antagonist Activity in Constitutively Active
    TABLE 1 Studies of antagonist activity in constitutively active receptors systems shown to demonstrate inverse agonism for at least one ligand Targets are natural Gs and constitutively active mutants (CAM) of GPCRs. Of 380 antagonists, 85% of the ligands demonstrate inverse agonism. Receptor Neutral Antagonist Inverse Agonist Reference Human β2-adrenergic Dichloroisoproterenol, pindolol, labetolol, timolol, Chidiac et al., 1996; Azzi et alprenolol, propranolol, ICI 118,551, cyanopindolol al., 2001 Turkey erythrocyte β-adrenergic Propranolol, pindolol Gotze et al., 1994 Human β2-adrenergic (CAM) Propranolol Betaxolol, ICI 118,551, sotalol, timolol Samama et al., 1994; Stevens and Milligan, 1998 Human/guinea pig β1-adrenergic Atenolol, propranolol Mewes et al., 1993 Human β1-adrenergic Carvedilol CGP20712A, metoprolol, bisoprolol Engelhardt et al., 2001 Rat α2D-adrenergic Rauwolscine, yohimbine, WB 4101, idazoxan, Tian et al., 1994 phentolamine, Human α2A-adrenergic Napthazoline, Rauwolscine, idazoxan, altipamezole, levomedetomidine, Jansson et al., 1998; Pauwels MPV-2088 (–)RX811059, RX 831003 et al., 2002 Human α2C-adrenergic RX821002, yohimbine Cayla et al., 1999 Human α2D-adrenergic Prazosin McCune et al., 2000 Rat α2-adrenoceptor MK912 RX821002 Murrin et al., 2000 Porcine α2A adrenoceptor (CAM- Idazoxan Rauwolscine, yohimbine, RX821002, MK912, Wade et al., 2001 T373K) phentolamine Human α2A-adrenoceptor (CAM) Dexefaroxan, (+)RX811059, (–)RX811059, RS15385, yohimbine, Pauwels et al., 2000 atipamezole fluparoxan, WB 4101 Hamster α1B-adrenergic
    [Show full text]
  • 1 Title Page Pharmacological Comparison of Mitragynine and 7
    JPET Fast Forward. Published on December 31, 2020 as DOI: 10.1124/jpet.120.000189 This article has not been copyedited and formatted. The final version may differ from this version. JPET-AR-2020-000189R1: Obeng et al. Pharmacological Comparison of Mitragynine and 7-Hydroxymitragynine: In Vitro Affinity and Efficacy for Mu-Opioid Receptor and Morphine-Like Discriminative-Stimulus Effects in Rats. Title Page Pharmacological Comparison of Mitragynine and 7-Hydroxymitragynine: In Vitro Affinity and Efficacy for Mu-Opioid Receptor and Opioid-Like Behavioral Effects in Rats Samuel Obeng1,2,#, Jenny L. Wilkerson1,#, Francisco León2, Morgan E. Reeves1, Luis F. Restrepo1, Lea R. Gamez-Jimenez1, Avi Patel1, Anna E. Pennington1, Victoria A. Taylor1, Nicholas P. Ho1, Tobias Braun1, John D. Fortner2, Morgan L. Crowley2, Morgan R. Williamson1, Victoria L.C. Pallares1, Marco Mottinelli2, Carolina Lopera-Londoño2, Christopher R. McCurdy2, Lance R. McMahon1, and Takato Hiranita1* Downloaded from Departments of Pharmacodynamics1 and Medicinal Chemistry2, College of Pharmacy, University of Florida jpet.aspetjournals.org #These authors contributed equally Recommended section: Behavioral Pharmacology at ASPET Journals on September 29, 2021 Correspondence: *Takato Hiranita, Ph.D. Department of Pharmacodynamics, College of Pharmacy, University of Florida 1345 Center Drive, Gainesville, FL 32610 Email: [email protected] Phone: +1.352.294.5411 Fax: +1.352.273.7705 Keywords: kratom, mitragynine, 7-hydroxymitragynine, morphine, opioid, discriminative stimulus Conflicts of Interests or Disclaimers: None Manuscript statistics: Number of text pages: 56 Number of tables: 7 Number of figures: 10 Number of supplementary Tables: 4 Number of supplementary figures: 7 1 JPET Fast Forward. Published on December 31, 2020 as DOI: 10.1124/jpet.120.000189 This article has not been copyedited and formatted.
    [Show full text]
  • Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation
    www.sciencemag.org/cgi/content/full/327/5963/348/DC1 Supporting Online Material for Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation Jason Rihel,* David A. Prober, Anthony Arvanites, Kelvin Lam, Steven Zimmerman, Sumin Jang, Stephen J. Haggarty, David Kokel, Lee L. Rubin, Randall T. Peterson, Alexander F. Schier* *To whom correspondence should be addressed. E-mail: [email protected] (A.F.S.); [email protected] (J.R.) Published 15 January 2010, Science 327, 348 (2010) DOI: 10.1126/science.1183090 This PDF file includes: Materials and Methods SOM Text Figs. S1 to S18 Table S1 References Supporting Online Material Table of Contents Materials and Methods, pages 2-4 Supplemental Text 1-7, pages 5-10 Text 1. Psychotropic Drug Discovery, page 5 Text 2. Dose, pages 5-6 Text 3. Therapeutic Classes of Drugs Induce Correlated Behaviors, page 6 Text 4. Polypharmacology, pages 6-7 Text 5. Pharmacological Conservation, pages 7-9 Text 6. Non-overlapping Regulation of Rest/Wake States, page 9 Text 7. High Throughput Behavioral Screening in Practice, page 10 Supplemental Figure Legends, pages 11-14 Figure S1. Expanded hierarchical clustering analysis, pages 15-18 Figure S2. Hierarchical and k-means clustering yield similar cluster architectures, page 19 Figure S3. Expanded k-means clustergram, pages 20-23 Figure S4. Behavioral fingerprints are stable across a range of doses, page 24 Figure S5. Compounds that share biological targets have highly correlated behavioral fingerprints, page 25 Figure S6. Examples of compounds that share biological targets and/or structural similarity that give similar behavioral profiles, page 26 Figure S7.
    [Show full text]
  • In Vivoactivation of a Mutantμ-Opioid Receptor by Naltrexone Produces A
    The Journal of Neuroscience, March 23, 2005 • 25(12):3229–3233 • 3229 Brief Communication In Vivo Activation of a Mutant ␮-Opioid Receptor by Naltrexone Produces a Potent Analgesic Effect But No Tolerance: Role of ␮-Receptor Activation and ␦-Receptor Blockade in Morphine Tolerance Sabita Roy, Xiaohong Guo, Jennifer Kelschenbach, Yuxiu Liu, and Horace H. Loh Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455 Opioid analgesics are the standard therapeutic agents for the treatment of pain, but their prolonged use is limited because of the development of tolerance and dependence. Recently, we reported the development of a ␮-opioid receptor knock-in (KI) mouse in which the ␮-opioid receptor was replaced by a mutant receptor (S196A) using a homologous recombination gene-targeting strategy. In these animals, the opioid antagonist naltrexone elicited antinociceptive effects similar to those of partial agonists acting in wild-type (WT) mice; however, development of tolerance and physical dependence were greatly reduced. In this study, we test the hypothesis that the failure of naltrexone to produce tolerance in these KI mice is attributable to its simultaneous inhibition of ␦-opioid receptors and activation of ␮-opioid receptors. Simultaneous implantation of a morphine pellet and continuous infusion of the ␦-opioid receptor antagonist naltrindole prevented tolerance development to morphine in both WT and KI animals. Moreover, administration of SNC-80 [(ϩ)-4-[(␣R)-␣-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], a ␦ agonist, in the naltrexone- pelleted KI animals resulted in a dose-dependent induction in tolerance development to both morphine- and naltrexone-induced anal- gesia. We conclude that although simultaneous activation of both ␮- and ␦-opioid receptors results in tolerance development, ␮-opioid receptor activation in conjunction with ␦-opioid receptor blockade significantly attenuates the development of tolerance.
    [Show full text]
  • Opioid Receptorsreceptors
    OPIOIDOPIOID RECEPTORSRECEPTORS defined or “classical” types of opioid receptor µ,dk and . Alistair Corbett, Sandy McKnight and Graeme Genes encoding for these receptors have been cloned.5, Henderson 6,7,8 More recently, cDNA encoding an “orphan” receptor Dr Alistair Corbett is Lecturer in the School of was identified which has a high degree of homology to Biological and Biomedical Sciences, Glasgow the “classical” opioid receptors; on structural grounds Caledonian University, Cowcaddens Road, this receptor is an opioid receptor and has been named Glasgow G4 0BA, UK. ORL (opioid receptor-like).9 As would be predicted from 1 Dr Sandy McKnight is Associate Director, Parke- their known abilities to couple through pertussis toxin- Davis Neuroscience Research Centre, sensitive G-proteins, all of the cloned opioid receptors Cambridge University Forvie Site, Robinson possess the same general structure of an extracellular Way, Cambridge CB2 2QB, UK. N-terminal region, seven transmembrane domains and Professor Graeme Henderson is Professor of intracellular C-terminal tail structure. There is Pharmacology and Head of Department, pharmacological evidence for subtypes of each Department of Pharmacology, School of Medical receptor and other types of novel, less well- Sciences, University of Bristol, University Walk, characterised opioid receptors,eliz , , , , have also been Bristol BS8 1TD, UK. postulated. Thes -receptor, however, is no longer regarded as an opioid receptor. Introduction Receptor Subtypes Preparations of the opium poppy papaver somniferum m-Receptor subtypes have been used for many hundreds of years to relieve The MOR-1 gene, encoding for one form of them - pain. In 1803, Sertürner isolated a crystalline sample of receptor, shows approximately 50-70% homology to the main constituent alkaloid, morphine, which was later shown to be almost entirely responsible for the the genes encoding for thedk -(DOR-1), -(KOR-1) and orphan (ORL ) receptors.
    [Show full text]
  • 4 Supplementary File
    Supplemental Material for High-throughput screening discovers anti-fibrotic properties of Haloperidol by hindering myofibroblast activation Michael Rehman1, Simone Vodret1, Luca Braga2, Corrado Guarnaccia3, Fulvio Celsi4, Giulia Rossetti5, Valentina Martinelli2, Tiziana Battini1, Carlin Long2, Kristina Vukusic1, Tea Kocijan1, Chiara Collesi2,6, Nadja Ring1, Natasa Skoko3, Mauro Giacca2,6, Giannino Del Sal7,8, Marco Confalonieri6, Marcello Raspa9, Alessandro Marcello10, Michael P. Myers11, Sergio Crovella3, Paolo Carloni5, Serena Zacchigna1,6 1Cardiovascular Biology, 2Molecular Medicine, 3Biotechnology Development, 10Molecular Virology, and 11Protein Networks Laboratories, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, 34149, Trieste, Italy 4Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste, Italy 5Computational Biomedicine Section, Institute of Advanced Simulation IAS-5 and Institute of Neuroscience and Medicine INM-9, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany 6Department of Medical, Surgical and Health Sciences, University of Trieste, 34149 Trieste, Italy 7National Laboratory CIB, Area Science Park Padriciano, Trieste, 34149, Italy 8Department of Life Sciences, University of Trieste, Trieste, 34127, Italy 9Consiglio Nazionale delle Ricerche (IBCN), CNR-Campus International Development (EMMA- INFRAFRONTIER-IMPC), Rome, Italy This PDF file includes: Supplementary Methods Supplementary References Supplementary Figures with legends 1 – 18 Supplementary Tables with legends 1 – 5 Supplementary Movie legends 1, 2 Supplementary Methods Cell culture Primary murine fibroblasts were isolated from skin, lung, kidney and hearts of adult CD1, C57BL/6 or aSMA-RFP/COLL-EGFP mice (1) by mechanical and enzymatic tissue digestion. Briefly, tissue was chopped in small chunks that were digested using a mixture of enzymes (Miltenyi Biotec, 130- 098-305) for 1 hour at 37°C with mechanical dissociation followed by filtration through a 70 µm cell strainer and centrifugation.
    [Show full text]
  • Constitutive Activity of the Δ-Opioid Receptor Expressed in C6 Glioma Cells
    British Journal of Pharmacology (1999) 128, 556 ± 562 ã 1999 Stockton Press All rights reserved 0007 ± 1188/99 $15.00 http://www.stockton-press.co.uk/bjp Constitutive activity of the d-opioid receptor expressed in C6 glioma cells: identi®cation of non-peptide d-inverse agonists 1Claire L. Neilan, 3Huda Akil, 1,2James H. Woods & *,1John R. Traynor 1Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, Ann Arbor, Michigan, MI 48109-0632, USA; 2Department of Psychology, University of Michigan Medical School, 1301 MSRB III, Ann Arbor, Michigan, MI 48109-0632, USA and 3Mental Health Research Institute, University of Michigan Medical School, Ann Arbor, Michigan, MI 48109-0720, USA 1 G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned d-(OP1) receptor (C6d), or the cloned m-(OP3) opioid receptor (C6m). 2 Constitutive activity was measured in the absence of Na+ ions to provide an increased signal. The degree of constitutive activity was de®ned as the level of [35S]-GTPgS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6d cells the level of basal [35S]-GTPgS binding was reduced by 51.9+6.1 fmols mg71 protein, whereas in C6m and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPgS binding by only 10.0+3.5 and 8.6+3.1 fmols mg71 protein respectively. 3 The d-antagonists N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as d-opioid receptor inverse agonists.
    [Show full text]
  • A 0.70% E 0.80% Is 0.90%
    US 20080317666A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0317666 A1 Fattal et al. (43) Pub. Date: Dec. 25, 2008 (54) COLONIC DELIVERY OF ACTIVE AGENTS Publication Classification (51) Int. Cl. (76) Inventors: Elias Fattal, Paris (FR); Antoine A6IR 9/00 (2006.01) Andremont, Malakoff (FR); A61R 49/00 (2006.01) Patrick Couvreur, A6II 5L/12 (2006.01) Villebon-sur-Yvette (FR); Sandrine A6IPI/00 (2006.01) Bourgeois, Lyon (FR) (52) U.S. Cl. .......................... 424/1.11; 424/423; 424/9.1 (57) ABSTRACT Correspondence Address: Drug delivery devices that are orally administered, and that David S. Bradlin release active ingredients in the colon, are disclosed. In one Womble Carlyle Sandridge & Rice embodiment, the active ingredients are those that inactivate P.O.BOX 7037 antibiotics, such as macrollides, quinolones and beta-lactam Atlanta, GA 30359-0037 (US) containing antibiotics. One example of a Suitable active agent is an enzyme Such as beta-lactamases. In another embodi ment, the active agents are those that specifically treat colonic (21) Appl. No.: 11/628,832 disorders, such as Chrohn's Disease, irritable bowel syn drome, ulcerative colitis, colorectal cancer or constipation. (22) PCT Filed: Feb. 9, 2006 The drug delivery devices are in the form of beads of pectin, crosslinked with calcium and reticulated with polyethylene imine. The high crosslink density of the polyethyleneimine is (86). PCT No.: PCT/GBO6/OO448 believed to stabilize the pectin beads for a sufficient amount of time such that a Substantial amount of the active ingredi S371 (c)(1), ents can be administered directly to the colon.
    [Show full text]
  • Heterodimerization of Μ and Δ Opioid Receptors: a Role in Opiate Synergy
    The Journal of Neuroscience, 2000, Vol. 20 RC110 1of5 Heterodimerization of ␮ and ␦ Opioid Receptors: A Role in Opiate Synergy I. Gomes, B. A. Jordan, A. Gupta, N. Trapaidze, V. Nagy, and L. A. Devi Departments of Pharmacology and Anesthesiology, New York University School of Medicine, New York, New York 10016 Opiate analgesics are widely used in the treatment of severe ␮-selective ligands results in a significant increase in the bind- pain. Because of their importance in therapy, different strate- ing of a ␦ receptor agonist. This robust increase is also seen in gies have been considered for making opiates more effective SKNSH cells that endogenously express both ␮ and ␦ recep- while curbing their liability to be abused. Although most opiates tors. Furthermore, we find that a ␦ receptor antagonist en- exert their analgesic effects primarily via ␮ opioid receptors, a hances both the potency and efficacy of the ␮ receptor signal- number of studies have shown that ␦ receptor-selective drugs ing; likewise a ␮ antagonist enhances the potency and efficacy can enhance their potency. The molecular basis for these find- of the ␦ receptor signaling. A combination of agonists (␮ and ␦ ings has not been elucidated previously. In the present study, receptor selective) also synergistically binds and potentiates we examined whether heterodimerization of ␮ and ␦ receptors signaling by activating the ␮–␦ heterodimer. Taken together, could account for the cross-modulation previously observed these studies show that heterodimers exhibit distinct ligand between these two receptors. We find that co-expression of ␮ binding and signaling characteristics. These findings have im- and ␦ receptors in heterologous cells followed by selective portant clinical ramifications and may provide new foundations immunoprecipitation results in the isolation of ␮–␦ het- for more effective therapies.
    [Show full text]
  • Blockade of O-Opioid Receptors Prevents Morphine-Induced Place Preference in Mice
    Blockade of o-Opioid Receptors Prevents Morphine-Induced Place Preference in Mice Tsutomu Suzuki', Michiharu Yoshiike', Hirokazu Mizoguchi', Junzo Kamei', Miwa Misawa' and Hiroshi Nagase2 'Department of Pharmacology , School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142, Japan 2Basic Research Laboratories , Toray Industries, Inc., 111 Tebiro, Kamakura 248, Japan Received May 12, 1994 Accepted June 18, 1994 ABSTRACT-Effects of highly selective 5-opioid receptor antagonists on the morphine-induced place preference in ddY and p,-opioid receptor deficient CXBK mice were investigated. Pretreatment with naltrin dole (NTI: a non-selective 5-opioid receptor antagonist), 7-benzylidenenaltrexone (BNTX: a selective 5, opioid receptor antagonist) or naltriben (NTB: a selective 52-opioid receptor antagonist) abolished the mor phine-induced place preference in ddY mice in a dose-dependent manner. These findings suggest that the morphine-induced place preference may be mediated by both d, and 52-opioid receptors. On the other hand, in p,-opioid receptor deficient CXBK mice, pretreatment with these selective 5-opioid receptor an tagonists did not affect the morphine-induced place preference, although pretreatment with ;3-funaltrex amine (13-FNA: a selective p-opioid receptor antagonist) significantly inhibited the morphine-induced place preference. [D-Pen 2,D-Pen5]enkephalin (DPDPE: a 0,-opioid receptor agonist) and [D-Ala2, Glu4]deltorphin (deltorphin II: a 52-opioid receptor agonist) induced a significant place preference in ddY mice, but not in CXBK mice. These results suggest that d, and 52-opioid receptors in the nucleus accumbens that are related to the DPDPE and deltorphin II-induced place preference may be dysfunctional and/or poor in CXBK mice.
    [Show full text]
  • NIDA Drug Supply Program Catalog, 25Th Edition
    RESEARCH RESOURCES DRUG SUPPLY PROGRAM CATALOG 25TH EDITION MAY 2016 CHEMISTRY AND PHARMACEUTICS BRANCH DIVISION OF THERAPEUTICS AND MEDICAL CONSEQUENCES NATIONAL INSTITUTE ON DRUG ABUSE NATIONAL INSTITUTES OF HEALTH DEPARTMENT OF HEALTH AND HUMAN SERVICES 6001 EXECUTIVE BOULEVARD ROCKVILLE, MARYLAND 20852 160524 On the cover: CPK rendering of nalfurafine. TABLE OF CONTENTS A. Introduction ................................................................................................1 B. NIDA Drug Supply Program (DSP) Ordering Guidelines ..........................3 C. Drug Request Checklist .............................................................................8 D. Sample DEA Order Form 222 ....................................................................9 E. Supply & Analysis of Standard Solutions of Δ9-THC ..............................10 F. Alternate Sources for Peptides ...............................................................11 G. Instructions for Analytical Services .........................................................12 H. X-Ray Diffraction Analysis of Compounds .............................................13 I. Nicotine Research Cigarettes Drug Supply Program .............................16 J. Ordering Guidelines for Nicotine Research Cigarettes (NRCs)..............18 K. Ordering Guidelines for Marijuana and Marijuana Cigarettes ................21 L. Important Addresses, Telephone & Fax Numbers ..................................24 M. Available Drugs, Compounds, and Dosage Forms ..............................25
    [Show full text]