PC2 Null Mice Show Up-Regulation of the Mu Opioid Receptor in Key Brain Regions Related to Nociceptive Information and Addictive Behaviors

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 142-166-Hypothalamus-Pituitary Development & Biology
Basic/Clinical
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-152
Desean Lovell Lee*1, Kabir Lutfy2, Monica Gabriela Ferrini3 and Theodore C Friedman1
1Charles R. Drew University of Medicine and Science, Los Angeles, CA, 2Charles Drew University, 3Charles Drew University, Los Angeles, CA
Licit and illicit drug use and abuse are growing problems in the society. Drug addiction is a disease of the brain that results, in part, in the altered endogenous opioid system levels by exogenous agonists. Morphine is an exogenous opiate agonist that binds to the mu opioid receptor (MOR) and is addictive. Prohormone Convertase 2 (PC2) converts intermediate pro-hormones to active hormones in key brain regions. PC2 null mice show enhanced morphine-induced antinociception that might be due to low levels of endogenous opioids leading to up-regulation of the MOR. In these experiments, we assessed the level of mu opioid receptor in brain regions relevant to analgesia, reward and addictive behaviors in PC2 knock-out (KO) mice using immunohistochemistry. Our results showed an up-regulation of the MOR in Pere Periaqueductal Grey Area (PAG), Ventral  Tagmental Area (VTA), Lateral Hypothalamus (LH), Medial Hypothalamus (HT), Nucleus Accumbens (NA), and Somatosensorial Cortex (SSC) regions, with the highest up-regulation in the PAG and SSC regions respectfully (P>0.0005). We conclude that PC2 novelly regulates MOR expression in key brain areas related to pain and drug addiction and may play a role in determining the mechanism of transition from opioid drug use to abuse.

Literature Cited 1.         Marquez P, Baliram R, Gajawada N, Friedman TC, Lutfy K 2006 Differential involvement of enkephalins in analgesic tolerance, locomotor sensitization, and conditioned place preference induced by morphine. Behav Neurosci 120:10-15. 2.         Yoburn BC, Sierra V, Lutfy K 1989 Chronic opioid antagonist treatment: assessment of receptor upregulation. Eur J Pharmacol 170:193-200. 3.         Leshner AI 1997 Addiction is a brain disease, and it matters. Science 278:45-47 4.         Rodriguez de Fonseca F, Carrera M, Navarro M, Koob GF, Weiss F 1997 Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science 276:2050-2054 5.         Zhou Y, Spangler R, LaForge KS, Maggos CE, Ho A, Kreek MJ 1996 Corticotropin-releasing factor and type 1 corticotropin-releasing factor receptor messenger RNAs in rat brain and pituitary during "binge"-pattern cocaine administration and chronic withdrawal. J Pharmacol Exp Ther 279:351-358 6.         Spangler R, Zhou Y, Maggos CE, Zlobin A, Ho A, Kreek MJ 1996 Dopamine antagonist and "binge' cocaine effects on rat opioid and dopamine transporter mRNAs. Neuroreport 7:2196-2200 7.         Garcia de Yebenes E, Pelletier G 1993 Opioid regulation of proopiomelanocortin (POMC) gene expression in the rat brain as studied by in situ hybridization. Neuropeptides 25:91-94 8.         Harrison LM, Kastin AJ, Zadina JE 1998 Opiate tolerance and dependence: receptors, G-proteins, and antiopiates. Peptides 19:1603-1630 9.         Elshourbagy NA, Ames RS, Fitzgerald LR, Foley JJ, Chambers JK, Szekeres PG, Evans NA, Schmidt DB, Buckley PT, Dytko GM, Murdock PR, Milligan G, Groarke DA, Tan KB, Shabon U, Nuthulaganti P, Wang DY, Wilson S, Bergsma DJ, Sarau HM 2000 Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. J Biol Chem 275:25965-25971 10.       Boundy VA, Gold SJ, Messer CJ, Chen J, Son JH, Joh TH, Nestler EJ 1998 Regulation of tyrosine hydroxylase promoter activity by chronic morphine in TH9.0-LacZ transgenic mice. J Neurosci 18:9989-9995 11.       Erdtmann-Vourliotis M, Mayer P, Riechert U, Grecksch G, Hollt V 1998 Identification of brain regions that are markedly activated by morphine in tolerant but not in naive rats. Brain Res Mol Brain Res 61:51-61 12.       Lane-Ladd SB, Pineda J, Boundy VA, Pfeuffer T, Krupinski J, Aghajanian GK, Nestler EJ 1997 CREB (cAMP response element-binding protein) in the locus coeruleus: biochemical, physiological, and behavioral evidence for a role in opiate dependence. J Neurosci 17:7890-7901 13.       Loh HH, Tseng LF, Wei E, Li CH 1976 Beta-endorphin is a potent analgesic agent. Proc Natl Acad Sci USA 73:2895-2898 14.       Cowan A, Murray CW 1990 Effect of norbinaltorphimine on the behavoir of mice and rats receiving multiple injections of U-50,488H. Prog Clin Biol Res 328:303-306 15.       Bare LA, Mansson E, Yang D 1994 Expression of two variants of the human mu opioid receptor mRNA in SK-N-SH cells and human brain. FEBS Lett 354:213-216 16.       Zimprich A, Simon T, Hollt V 1995 Cloning and expression of an isoform of the rat mu opioid receptor (rMOR1B) which differs in agonist induced desensitization from rMOR1. FEBS Lett 359:142-146 17.       Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL 1996 Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383:819-823 18.       Sora I, Takahashi N, Funada M, Ujike H, Revay RS, Donovan DM, Miner LL, Uhl GR 1997 Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia. Proc Natl Acad Sci U S A 94:1544-1549 19.       Schuller AG, King MA, Zhang J, Bolan E, Pan YX, Morgan DJ, Chang A, Czick ME, Unterwald EM, Pasternak GW, Pintar JE 1999 Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1. Nat Neurosci 2:151-156 20.       Bronstein DM, Przewlocki R, Akil H 1990 Effects of morphine treatment on pro-opiomelanocortin systems in rat brain. Brain Res 519:102-111 21.       Gudehithlu K, Tejwani GA, Bhargava HN 1991 b-Endorphin and methionine-encephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats. Brain Res 553:284-290 22.       Przewlocki R, Hollt V, Duka TH, Kleber G, Gramsch CH, Haarmann I, Herz A 1979 Long-term morphine treatment decreases endorphin levels in rat brain and pituitary. Brain Res 174:357-361 23.       Bronstein DM, Day NC, Gutstein HB, Trujillo KA, Akil H 1993 Pre- and posttranslational regulation of b-endorphin biosynthesis in the CNS: effects of chronic naltrexone treatment. J Neurochem 60:40-49 24.       Clement-Jones V, McLoughlin L, Lowry LP, Besser GM, Rees lH 1979 Acupuncture in heroin addicts; changes in met-enkephalin and endorphin in blood and cerebrospinal fluid. Lancet 2:380-383 25.       Terenius LT, O'Brien CP 1992 Receptors and endogenous ligands: implications for addiction. In: O'Brien CP, Jaffe JH eds. Addictive states. New York: Raven Press, Ltd.; 123-130 26.       Blum K 1983 Alcohol and central nervous system peptides. Subst and Alc Actions/Misuse 4:73-87 27.       Erickson CK 1990 Reviews and comments on alcohol research. Alcohol 7:557-558 28.       Volpicelli JR, Davis MA, Olgin JE 1986 Naltrexone blocks the post-shock increase of ethanol consumption. Life Sci 38:841-847 29.       Rubinstein M, Mogil JS, Japon M, Chan EC, Allen RG, Low MJ 1996 Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis. Proc Natl Acad Sci USA 93:3995-4000 30.       Gianoulakis C, Drouin JN, Seidah NG, Kalant H, Chretien M 1981 Effect of chronic morphine treatment on beta-endorphin biosynthesis by the rat neurointermediate lobe. Eur J Pharmacol 72:313-321 31.       Seidah NG, Marcinkiewicz M, Benjannet S, Gaspar L, Beaubien G, Mattei MG, Lazure C, Mbikay M, Chretien M 1991 Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol 5:111-122 32.       Seidah NG, Gaspar L, Mion P, Marcinkiewicz M, Mbikay M, Chretien M 1990 cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA Cell Biol 9:415-424 33.       Smeekens SP, Steiner DF 1990 Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J Biol Chem 265:2997-3000 34.       Smeekens SP, Avruch AS, LaMendola J, Chan SJ, Steiner DF 1991 Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci USA 88:340-344 35.       Hakes DJ, Birch NP, Mezey E, Dixon JE 1991 Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats. Endocrinology 129:3053-3063 36.       Birch NP, Tracer HL, Hakes DJ, Loh YP 1991 Coordinate regulation of mRNA levels of pro-opiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in the rat pituitary intermediate lobe. Biochem Biophys Res Commun 179:1311-1319 37.       Seidah NG, Chretien M, Day R 1994 The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. Biochimie 76:197-209 38.       Estivariz FE, Friedman TC, Chikuma T, Loh YP 1992 Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. J Biol Chem 267:7456-7463 39.       Friedman TC, Cool DR, Jayasvasti V, Louie D, Loh YP 1996 Processing of pro-opiomelanocortin in GH3 cells: inhibition by prohormone convertase 2 (PC2) antisense mRNA. Mol Cell Endocrinol 116:89-96 40.       Friedman TC, Loh YP, Birch NP 1994 In vitro processing of proopiomelanocortin by recombinant PC1 (SPC3). Endocrinology 135:854-862 41.       Benjannet S, Rondeau N, Day R, Chretien M, Seidah NG 1991 PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 88:3564-3568 42.       Thomas L, Leduc R, Thorne BA, Smeekens SP, Steiner DF, Thomas G 1991 Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 88:5297-5301 43.       Seidah NG, Fournier H, Boileau G, Benjannet S, Rondeau N, Chretien M 1992 The cDNA structure of the porcine pro-hormone convertase PC2 and the comparative processing by PC1 and PC2 of the N-terminal glycopeptide segment of porcine POMC. FEBS Lett 310:235-239 44.       Bloomquist BT, Eipper BA, Mains RE 1991 Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA. Mol Endocrinol 5:2014-2024 45.       Johanning K, Juliano MA, Juliano L, Lazure C, Lamango NS, Steiner DF, Lindberg I 1998 Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates. J Biol Chem 273:22672-22680 46.       Johanning K, Mathis JP, Lindberg I 1996 Role of PC2 in proenkephalin processing: antisense and overexpression studies. J Neurochem 66:898-907 47.       Dupuy A, Lindberg I, Zhou Y, Akil H, Lazure C, Chretien M, Seidah NG, Day R 1994 Processing of prodynorphin by the prohormone convertase PC1 results in high molecular weight intermediate forms. Cleavage at a single arginine residue. FEBS Lett 337:60-65 48.       Day R, Lazure C, Basak A, Boudreault A, Limperis P, Dong W, Lindberg I 1998 Prodynorphin processing by proprotein convertase 2. Cleavage at single basic residues and enhanced processing in the presence of carboxypeptidase activity. J Biol Chem 273:829-836 49.       Furuta M, Carroll R, Martin S, Swift HH, Ravazzola M, Orci L, Steiner DF 1998 Incomplete processing of proinsulin to insulin accompanied by elevation of Des-31,32 proinsulin intermediates in islets of mice lacking active PC2. J Biol Chem 273:3431-3437 50.       Furuta M, Yano H, Zhou A, Rouille Y, Holst JJ, Carroll R, Ravazzola M, Orci L, Furuta H, Steiner DF 1997 Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci U S A 94:6646-6651 51.       Berman Y, Mzhavia N, Polonskaia A, Furuta M, Steiner DF, Pintar JE, Devi LA 2000 Defective prodynorphin processing in mice lacking prohormone convertase PC2. J Neurochem 75:1763-1770 52.       Allen RG, Peng B, Pellegrino MJ, Miller ED, Grandy DK, Lundblad JR, Washburn CL, Pintar JE 2001 Altered processing of pro-orphanin FQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone convertase 2. J Neurosci 21:5864-5870 53.       Allen RG, Pelligrino MJ, Peng B, Pintar JE 1999 Altered POMC processing in SPC2 KO mice. Endo Soc 81:P1-640 (abstract) 54.       Laurent V, Jaubert-Miazza L, Desjardins R, Day R, Lindberg I 2004 Biosynthesis of proopiomelanocortin-derived peptides in prohormone convertase 2 and 7B2 null mice. Endocrinology 145:519-528. Epub 2003 Oct 2023. 55.       Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, Laurent V, Lindberg I, Ugleholdt R, Holst JJ, Steiner DF 2002 Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc Natl Acad Sci U S A 99:10293–10298 56.       Zhu X, Orci L, Carroll R, Norrbom C, Ravazzola M, Steiner DF 2002 Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3. Proc Natl Acad Sci U S A 99:10299-10304 57.       Schroeder SA, Brandes N, Orleans CT, Kaufman N 1999 Thematic review series V: substance abuse research and clinical practice. Proc Assoc Am Physicians 111:97-98 58.       Rothman RB, Long JB, Bykov V, Xu H, Jacobson AE, Rice KC, Holaday JW. Upregulation of the opioid receptor complex by the chronic administration of morphine: a biochemical marker related to the development of tolerance and dependence. Peptides. 1991 Jan-Feb;12(1):151-60. Erratum in: Peptides 1991 May-Jun;12(3):671. PubMed PMID: 1646998. 59.          Ritter, I. Mocchetti, A., and E. Costa. 2008 "Down-regulation of Proopiomelanocortin Synthesis and Beta-endorphin Utilization in Hypothalamus of Morphine-tolerant Rats." Journal of Molecular Neuroscience 1.1 (2008): 33-38. SpringerLink. Springer Science Business Media, 30 Nov. 2008. Web. 23 Feb. 2012 60.       Leshner AI 1997 Addiction is a brain disease, and it matters. Science 278:45-47 62.       Fábián G, Bozó B, Szikszay M, Horváth G, Coscia CJ, Szücs M. 2002 Chronic morphine-induced changes in mu-opioid receptors and G proteins of different subcellular loci in rat brain. J Pharmacol Exp Ther. 2002 Aug;302(2):774-80 63.       Shah YB, Haynes L, Prior MJ, Marsden CA, Morris PG, Chapman V. 2005 Functional magnetic resonance imaging studies of opioid receptor-mediated modulation of noxious-evoked BOLD contrast in rats. Psychopharmacology (Berl). 2005 Aug;180(4):761-73. Epub 2005 Sep 14. Bozarth MA (1987a). An overview of assessing drug reinforcement. In: Bozarth MA (ed) Methods of assessing the reinforcing properties of abuse drugs (pp. 635-658), Springer-Verlag, New York 64.       Furuta M, Yano H, Zhou A, Rouille Y, Holst JJ, Carroll R, Ravazzola M, Orci L, Furuta H, Steiner DF 1997 Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci U S A 94:6646-6651 65.       Espinosa VP, Liu Y, Ferrini M, Anghel A, Nie Y, Tripathi PV, Porche R, Jansen E, Stuart RC, Nillni EA, Lutfy K, Friedman TC. 2008 Differential regulation of prohormone convertase 1/3, prohormone convertase 2 and phosphorylated cyclic-AMP-response element binding protein by short-term and long-term morphine treatment: implications for understanding the "switch" to opiate addiction. Neuroscience. 2008 Oct 15;156(3):788-99. Epub 2008 Aug 9. 66.       Richard G. Allen, Bonnie Peng, Michael J. Pellegrino, Emilie D. Miller, David K. Grandy, James R. Lundblad, Carrie L. Washburn, and John E. Pintar. 2002 Altered Processing of Pro-Orphanin FQ/Nociceptin and Pro-Opiomelanoortin-Derived Peptides in the Brains of Mice Expressing Defective Prohormone Convertase 2. The Journal of Neuroscience, 15 August 2001, 21(16): 5864-5870

Nothing to Disclose: DLL, KL, MGF, TCF

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Research funded by  Minority Institutions' Drug Abuse Research Development Program (MIDARP) Grant.