Preface Antisense technology reached a watershed year in 1998 with the FDA approval of the antisense-based therapy, Vitravene, developed by ISIS. This is the first drug based on antisense technology to enter the marketplace and makes antisense technology a reality for therapeutic applications. How- ever, antisense technology still needs further development, and new applica- tions need to be explored. Contained in this Volume 314 (Part B) of Methods in Enzymology and its companion Volume 313 (Part A) are a wide range of methods and applications of antisense technology in current use. We set out to put together a single volume, but it became obvious that the variations in methods and the numerous applications required at least two volumes, and even these do not, by any means, cover the entire field. Nevertheless, the articles included represent the work of active research groups in industry and academia who have developed their own methods and techniques. In this volume, Part B: Applications, chapters cover methods in which anti- sense is designed to target membrane receptors and antisense application in the neurosciences, as well as in nonneuronal tissues. The therapeutic applications of antisense technology, the latest area of new interest, com- plete the volume. In Part A: General Methods, Methods of Delivery, and RNA Studies several methods of antisense design and construction are included as are general methods of delivery and antisense used in RNA studies. Although Methods in Enzymology is designed to emphasize methods, rather than achievements, I congratulate all the authors on their achieve- ments that have led them to make their methods available. In compiling and editing these two volumes I could not have made much progress without the excellent secretarial services of Ms. Gayle Butters of the University of Florida, Department of Physiology. M. IAN PHILLIPS XV Contributors to Volume 314 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. FE C. ABOGADIE (10), Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College Lon- don, London WC1E 6BT, United Kingdom YIJIA BAO (12), Vysis, Inc., Downers Grove, Illinois 60515 RUTH BEATTIE (21), Lilly Research Centre, Eli Lilly and Company Limited, Win- dlesham, Surrey GU20 6PH, United Kingdom MARGERY C. BEINFELD (8), Department of Pharmacology and Experimental Thera- peutics, Tufts University School of Medi- cine, Boston, Massachusetts 02111 GAETANO BERGAMASCHI (30), Dipartimento di Medicina Interna e Terapia Medica, Medicina Interna e Oncologia Medica, I.R.C.C.S. Policlinico San Matteo, 27100 Pavia, Italy E. A. L. BIESSEN (23), Division of Biophar- maceutics, Leiden~Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands M. K. BIJSTERBOSCH (23), Division of Bio- pharmaceutics, Leiden~Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands H. E. BLUM (37), Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany FR/~DI~RIC BOST (24), Sidney Kimmel Cancer Center, San Diego, California 92121 DUSTIN H. O. BRITrON (12), DuPont Pharma- ceuticals, Wilmington, Delaware 19880 WILLIAM C. BROADDUS (9), Division of Neu- rosurgery and Department of Anatomy, Medical College of Virginia, Virginia Com- monwealth University, Richmond, Virginia 23298-0631 DAVID A. BROWN (10), Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College Lon- don, London WC1E 6BT, United Kingdom NOEL J. BUCKLEY (10), School of Biochemis- try and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom MARINA CATSICAS (11), Department of Physi- ology, University College London, London, United Kingdom STEFAN CATSICAS (11), Institut de Biologie Cellulaire et de Morphologie, Universit~ de Lausanne, CH-I O05 Lausanne, Switzerland MALCOLM P. CAULFIELD (10), Wellcome Lab- oratory for Molecular Pharmacology, Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom MARIO CAZZOLA (30), Dipartimento di Medi- cina Interna e Terapia Medica, Medicina Interna e Oncologia Medica, I.R.C.C.S. Policlinico San Matteo, 27100 Pavia, Italy BYLrNG-MIN CHOl (34), Department of Micro- biology and Immunology, Wonkwang Uni- versity School of Medicine, Iksan-shi, Chonbug 570-749, Korea CHUAN-CHu CHOU (29), Department of Pa- thology, New Jersey Medical School, Uni- versity of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103 HuN-TAEG CHUNG (34), Department of Mi- crobiology and Immunology, Wonkwang University School of Medicine and Medici- nal Resources Research Center of Wonk- wang University, Iksan-shi, Chonbug 570- 749, Korea CATHERINE L. CIOFF1 (25), Department of Metabolic and Cardiovascular Diseases, Novartis Institute for Biomedical Research, Summit, New Jersey 07901 ix X CONTRIBUTORS TO VOLUME 314 PETER J. CRAIG (21), Lilly Research Centre, Eli Lilly and Company Limited, Win- dlesham, Surrey GU20 6PH, United Kingdom MONICA CURTO (18), Department of Cyto- morphology, School of Medicine, Cittadella University, Cagliari, Italy MARIZA DAYRELL (10), Wellcome Labora- tory for Molecular Pharmacology, Depart- ment of Pharmacology, University College London, London WC1E 6BT, United Kingdom NICHOLAS M. DEAN (24), ISIS Pharmaceuti- cals, Inc., Carlsbad, California 92008 ISABEL DE ANTONIO (1), Department of Neu- ropathology, Cajal Institute, Consejo Supe- rior de Investigaci6nes Cientificas, E-28002 Madrid, Spain PATRICK DELMAS (10), Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College Lon- don, London WC1E 6BT, United Kingdom MICHEL DE WAARD (21), Laboratoire de Neu- robiologie des Canaux Ioniques, INSERM U374, 13916 Marseille Cedex 20, France MICHAEL G. DUBE (13), Department of Physi- ology, University of Florida Brain Institute, University of Florida College of Medicine, Gainesville, Florida 32610-0274 MARCEL EGGER (32), Department of Physiol- ogy, University of Bern, CH-3012 Bern, Switzerland ANNE FELTZ (21), Laboratoire de Neuro- biologie Cellulaire, UPR 9009 Centre National de la Recherche Scientifique, 67084 Strasbourg, France HELEN L. FILLMORE (9), Division of Neu- rosurgery, Medical College of Virginia, Virginia Commonwealth University, Rich- mond, Virginia 23298-0631 K. FLUITER (23), Division of Biopharma- ceutics, Leiden~Amsterdam Center for Drug Research, Leiden University, 2360 RA Leiden, The Netherlands MICHAEL B. GANZ (26), Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 JAVIER GARZ6N (1), Department of Neuropa- thology, Cajal Institute, Consejo Superior de Investigaci6nes Cientificas, E-28002 Madrid, Spain SAMANTHA GILLARD (21), Department of Pharmacology, University of Chicago, Chi- cago, Illinois 60637 GEORGE T. GILLIES (9), Department of Bio- medical Engineering, Health Sciences Cen- ter, University of Virginia, Charlottesville, Virginia 22908 SUSAN GOULD-FOGERITE (29), Department of Pathology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103 MARIA GRAZIA ENNAS (18), Department of Cytomorphology, School of Medicine, Cittadella University, Cagliari, Italy FULVIA GREMO (18), Department of Cytomor- phology, School of Medicine, Cittadella University, Cagliari, Italy GABRIELE GRENNINGLOH (11), Institut de Biologie Cellulaire et de Morphologie, Universit~ de Lausanne, CH-IO05 Lau- sanne, Switzerland JANE E. HALEY (10), Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College Lon- don, London WC1E 6BT, United Kingdom FINN HALLB00K (11), Department of Neuro- science, Uppsala University, BMC, S-751 23 Uppsala, Sweden MAX HE (29), Department of Pathology, New Jersey Medical School, University of Medi- cine and Dentistry of New Jersey, Newark, New Jersey 07103 MATTHEW O. HEBB (19), Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8 MARKUS HEILIG (19), Department of Clinical Neuroscience, Occupational Therapy and Elderly Care Research, Karolinska Institute, Stockholm, Sweden JULIE G. HENSLER (6), Department of Phar- macology, University of Texas Health Sci- ence Center, San Antonio, Texas 78284- 7764 CONTRIBUTORS TO VOLUME 314 xi SIEW PENG HO (12), DuPont Pharmaceuti- cab, Wilmington, Delaware 19880 JEFFREY T. HOLT (35), Departments of Cell Biology and Pathology, Vanderbilt Univer- sity Medical School, Nashville, Tennessee 37232 ALAN L. HUDSON (5), Psychopharmacology Unit, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom JOHN C. HUNTER (14), Center for Biological Research, Roche Bioscience, Palo Alto, California 94304 ANTrI P. JEKUNEN (36), Department of Clini- cal Pharmacology, Helsinki University Cen- tral Hospital, FIN-O0029 HYKS, Finland ROY A. JENSEN (35), Departments of Pathol- ogy and Cell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232 MA~DALENA JUHASZOVA (22), Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201 C~IANG-DUK JUN (34), Department of Mi- crobiology and Immunology, Wonkwang University School of Medicine, lksan-shi, Chonbug 570-749, Korea KALEVI J. A. KAIREMO (36), Department of Clinical Chemistry, Norwegian University of Science and Technology, N-7006 Trond- heim, Norway PUSHPA S. KALRA (13), Department of Physi- ology, University of Florida Brain Institute, University of Florida College of Medicine, Gainesville, Florida 32611 SATVA P. KALRA (13), Department of Neuro- science, University of Florida Brain Insti- tute, University of Florida College of Medi- cine, Gainesville, Florida 32611 JESPER KARLE (2), Department of Psychiatry, Rigshospitalet (National Hospital), DK- 2100 Copenhagen, Denmark MICHAEL J. KATOVICH (39), Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, Florida 3261O JOSEPHINE LAI (14), Department of Pharma- cology, University of Arizona, Tucson, Ari- zona 85724 REGIS C. LAMBERT (21), Laboratoire de Neu- robiologie Cellulaire, UPR 9009 Centre Na- tional de la Recherche Scientifique, 67084 Strasbourg, France PETER LIPP (32), Laboratory of Molecular Signalling, The Babraham Institute, Cam- bridge CB2 3E J, United Kingdom DI Lu (39), Department of Physiology, Uni- versity of Florida College of Medicine, Gainesville, Florida 32610 CLAUDIA LUCOTI'I (30), Dipartimento di Medicina Interna e Terapia Medica, Medicina Interna e Oncologia Medica, I.R.C.C.S. Policlinico San MatteD, 27100 Pavia, Italy DAVID L. MATI'SON (27), Department of Phys- iology, Medical College of Wisconsin, Mil- waukee, Wisconsin 53226 YVES MAULET (21), Laboratoire de Neuro- biologie Cellulaire, UPR 9009 Centre Na- tional de la Recherche Scientifique, 67084 Strasbourg, France ROBERT McKAY (24), ISIS Pharmaceuticals, Inc., Carlsbad, California 92008 DAN MERCOLA (24), Sidney Kimmel Cancer Center, San Diego, California 92121, and Center for Molecular Genetics, University of California at San Diego, La JoUa, Cali- fornia 92093 RADMILA MILEUSNIC (15), Department of Bi- ological Sciences, The Open University, Milton Keynes MK7 6AA, United Kingdom DAGMARA MOHUCZY (3), Department of Physiology, University of Florida College of Medicine, Gainesville, Florida 32610 BRETr P. MONIA (25), Department of Molecu- lar Pharmacology, ISIS Pharmaceuticals, Inc., Carlsbad, California 92008 D. MORADPOUR (37), Department of Medi- cine II, University of Freiburg, D-79106 Freiburg, Germany xii COmmBUTORS TO VOLUME 314 ISABELLA MUSSINI (18), CNR Center of Mus- cle Biology and Physiopathology, Univer- sity of Padova, Padova, Italy INGA D. NEUMANN (16), Max Planck Institute of Psychiatry, D-80804 Munich, Germany MOGENS NIELSEN (2), Research Institute of Biological Psychiatry, St. Hans Hospital DK-4000 Roskilde, Denmark ERNST NIGGLI (32), Department of Physiol- ogy, University of Bern, CH-3012 Bern, Switzerland THADOEOS S. NOWAK, JR. (17), Departments of Anesthesiology and Resuscitology, Okayama University School of Medicine, Shikata-cho, Okayama City, Japan TAKAHIRO OCHIYA (28), Section for Studies on Metastasis, National Cancer Center Re- search Institute, Chuo-ku, Tokyo 104- 0045, Japan S. OFFENSPERGER (37), Department of Medi- cine II, University of Freiburg, D-79106 Freiburg, Germany WOLF-BERNHARD OFFENSPERGER (37), De- partment of Medicine II, University of Frei- burg, D-79106 Freiburg, Germany MICHAEL H. OssIvov (14), Department of Pharmacology, University of Arizona, Tuc- son, Arizona 85724 HYuN-OCK PAL (34), Department of Micro- biology and Immunology, Wonkwang Uni- versity. School of Medicine, Iksan-shi, Chonbug 570-749, Korea YING-XIAN PAN (4), Memorial Sloan-Ketter- ing Cancer Center, New York, New York 10021 GEORGE A. PARKER (29), Department of Pa- thology, New Jersey Medical School, Uni- versity of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103 GAVRm W. PASTERNAK (4), Memorial Sloan- Kettering Cancer Center, New York, New York 10021 BmAI PENG (29), Department of Pathology, New Jersey Medical School University of Medicine and Dentistry of New Jersey, New- ark, New Jersey 07103 M. IAN PHILLIPS (3), Department of Physiol- ogy, University of Florida College of Medi- cine, Gainesville, Florida 32610 FRANK PORRECA (14), Department of Phar- macology, University of Arizona, Tucson, Arizona 85724 O. POTAPOVA (24), Laboratory of Biological Chemistry, Gerontology Research Center, National Institute of Aging, National Insti- tute of Health, Baltimore, Maryland 21224 SUJIT S. PRABHU (9), Division of Neurosur- gery, Medical College of Virginia, Virginia Commonwealth University, Richmond, Vir- ginia 23298-0631 MOHAN K. RAIZADA (39), Department of Physiology, University of Florida College of Medicine, Gainesville, Florida 32610 ELIZABETH S. RAVECHI~ (29), Department of Pathology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103 PHYLLIS Y. REAVES (39), Department of Phys- iology, University of Florida College of Medicine, Gainesville, Florida 32610 EMMA S. J. ROBINSON (5), Psychopharma- cology Unit, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom CHERYL ROBINSON-BENION (35), Department of CeU Biology, Vanderbilt University Med- ical School, Nashville, Tennessee 37232 VITTORIO ROSTI (30), Laboratorio di Ricerca Area Trapianti, Unitd di lmmunologia Clin- ica, I.R.C.C.S. Policlinico San Matteo, 27100 Pavia, Italy E. T. RUMP (23), Division of Biopharmaceu- tics, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands PILAR S~NCHEZ-BL/~ZQUEZ (1), Department of Neuropathology, Cajal Institute, Consejo Superior de Investigaci6nes Cientificas, E-28002 Madrid, Spain JOANNE M. SCALZITTI (6), Department of Pharmacology, New York University Medi- cal School, New York, New York 10016 CONTRIBUTORS TO VOLUME 314 xiii CHRISTOPH SCHUMACHER (31), MCD Re- search, Novartis Pharmaceuticals Corpora- tion, Summit, New Jersey 07901-1398 BEAT SCHWALLER (32), Department of Histol- ogy and General Embryology, University of Fribourg, CH-1700 Fribourg, Switzerland MICHAEL S. SCULLY (12), DuPont Pharma- ceuticals, Wilmington, Delaware 19880 MARIELLA SETZU (18), Department of Cyto- morphology, School of Medicine, Cittadella University, Cagliari, Italy A. PAULA SIM(3Es-WUEST (33), Department of Internal Medicine, University Hospital Zurich, CH-8044 Zurich, Switzerland MARTIN K. SLODZ1NSKI (22), Department of Physiology, University of Maryland School of Medicine, and Department of Medicine, Mercy Hospital, Baltimore, Maryland 21201 JANET B. SMITH (38), Departments of Microbi- ology and Immunology, Kimmel Cancer Center, Cardeza Foundation for Hemato- logical Research, Thomas Jefferson Univer- sity, Philadelphia, Pennsylvania 19107-5083 VALERIA SOGOS (18), Department of Cyto- morphology, School of Medicine, Cittadella University, Cagliari, Italy DONG-HWAN SOHN (34), College of Phar- macy, Wonkwang University, Iksan-shi, Chonbug 570-749, Korea WOLFGANG SOMMER (19), Department of Clinical Neuroscience, Occupational Ther- apy and Elderly Care Research, Karolinska Institute, Stockholm, Sweden KELLY M. STANDIFER (7), Department of Pharmacological and Pharmaceutical Sci- ences, University of Houston, Houston, Texas 77204-5515 JULIE K. STAPLE (11), Institut de Biologie Cel- lulaire et de Morphologie, UniversiM de Lausanne, CH-I O05 Lausanne, Switzerland XIAOPING TANG (3), Department of Physiol- ogy, University of Florida College of Medi- cine, Gainesville, Florida 32610 MlraCo TENHUNEN (36), Department of On- cology, Helsinki University Central Hospi- tal, FIN-O0029 HYKS, Finland MASAAKI TERADA (28), National Cancer Cen- ter, Chuo-ku, Tokyo 104-0045, Japan C. THOMA (37), Department of Medicine, University of Freiburg, D-79106 Freiburg, Germany WOLFGANG TISCHMEYER (20), Leibniz Insti- tute for Neurobiology, D-39008 Magde- burg, Germany NICOLA TOSCHI (16), Max Planck Institute of Psychiatry, D-80804 Munich, Germany T. J. C. VAN BERKEL (23), Division of Bio- pharmaceutics, Leiden~Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands H. VIETSCH (23), Division of Biopharmaceu- tics, Leiden~Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands DAESETY VISHNUVARDHAN (8), Department of Pharmacology and Experimental Thera- peutics, Tufts University School of Medi- cine, Boston, Massachusetts 02111 STEPHEN G. VOLSEN (21), Lilly Research Centre, Eli Lilly and Company Limited, Windlesham, Surrey GU20 6PH, United Kingdom F. YON WEIZS.~CKER (37), Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany HONGWEI WANG (39), Department of Physiol- ogy, University of Florida College of Medi- cine, Gainesville, Florida 32610 ERIC WICKSTROM (38), Department of Micro- biology, Thomas Jefferson University, Phil- adelphia, Pennsylvania 19107 SUSANNA Wu-PoNG (9), Department of Pharmaceutics, Medical College of Vir- ginia, Virginia Commonwealth University, Richmond, Virginia 23298 YUTAKA YAIDA (17), Departments of Anes- thesiology and Resuscitology, Okayama University School of Medicine, Shikata-cho, Okayama City, Japan xiv CONTRIBUTORS TO VOLUME 314 JI-CHA/qG YOO (34), Department of Microbi- ology and Immunology, Wonkwang Uni- versity School of Medicine, Iksan-shi, Chonbug 570-749, Korea KATStJMI YUFU (17), Departments of Anesthe- siology and Resuscitology, Okayama Uni- versity School of Medicine, Shikata-cho, Okayama City, Japan UWE ZANGEMEISTER-WITI~E (33), Depart- ment of lnternal Medicine, University Hos- pital Zurich, CH-8044 Zurich, Switzerland ANNEMARIE ZIEGLER (33), Laboratory for Molecular and Cellular Oncology, Cancer Program, Faculty of Medicine, Catholic University of Chile, Santiago, Chile [1] ANTISENSE AND OPIOID RECEPTOR SIGNALING IN CNS 3 [I] In Vivo Modulation of G Proteins and Opioid Receptor Function by Antisense Oligodeoxynucleotides By JAVIER GARZ6N, ISABEL DE ANTONIO, and PILAR SANCHEZ-BLkZQUEZ Introduction Opioids promote their effects by acting on cellular receptors located in the cell membrane. The diversity of these receptors, which was initially suggested by pharmacological and biochemical studies, has been confirmed by the cloning of cDNAs encoding the/z, I'2 8~ 3'4 and w s types. The cloned opioid receptors have been found to correspond to the pharmacological subtypes tzl, 82, and K1. The subtypes ~2, 81, K2, and K3 (and possibly others) are still to be designated molecular identities. The use of antisense technology with the opioid system has been helpful in making molecular/ pharmacological correlations and the approach is currently being used to investigate uncorrelated subtypes. Opioid receptors couple to heterotrimeric (a,/3, and 3' subunits) GTP- binding regulatory proteins (G proteins). Our knowledge regarding the diversity and properties of G proteins has increased greatly. 6-s Most of the known classes are present in the nervous system and are thought to regulate various signaling pathways, e.g., adenylyl cyclases, different types of K + and Ca 2+ channels, phospholipases, protein kinases, and others. Ga subunits show differences in the regions involved in the interaction with membrane receptors (see, e.g., Jones and Reed9). These variations seem to account 1 y. Chen, A. Mestek, J. Liu, J. A. Hurley, and L. Yu, Mol. Pharmacol. 44, 8 (1993). 2 R. C. Thompson, A. Mansour, H. Akil, and S. J. Watson, Neuron 11, 903 (1993). 3 C. J. Evans, D. E. Keith, H. Morrison, K. Magendzo, and R. H. Edwards, Science 28, 1952 (1992). 4 B. L. Kieffer, K. Befort, C. Gaveriaux-Ruff, and C. G. Hirth, Proc. Natl. Acad. Sci. U.S.A. 89, 12048 (1992). 5 G. X. Xie, F. Meng, A. Mansour, R. C. Thompson, M. T. Hoversten, A. Goldstein, S. T. Watson, and H. Akil, Proc. Acad. Natl. Sci. U.S.A. 91, 3779 (1994). 6 n. E. Hamm and A. Gilchrist, Curr. Opin. Cell Biol. 8, 189 (1996). 7 E. J. Neer and T. F. Smith, Cell 84, 175 (1996). 8 D. G. Lambright, J. Sondek, A. Bohm, N. P. Skiba, H. E. Hamm, and P. B. Sigler, Nature (London) 379, 311 (1996). 9 D. T. Jones and R. R. Reed, J. Biol. Chem. 262, 14241 (1987). Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. METHODS IN ENZYMOLOGY, VOL. 314 0076-6879/99 $30.00 4 ANTISENSE RECEPTOR TARGETS [ 11 for the preference displayed by agonist-bound receptors to signal only via certain G proteins present in the cell membrane. 1°-13 Receptors also vary in the amino acid sequences that interact with ot subunits of trimeric G proteins, i.e., the receptor loop that links the fifth and sixth transmembrane region and the C-terminal tail. 14'15 The cloned ~- opioid receptor 3,4 and the /z-opioid receptor 1,2 differ in the intracellular territories implicated in their interaction with Ga subunits. It is conceivable, therefore, that distinct types or subtypes of opioid receptor regulate differ- ent classes of G proteins. 16-t8 Given the variations manifested by receptors and G proteins in their interacting domains, an investigation was made into the classes of G proteins regulated in vivo by/x and ~ receptors in the promotion of supraspinal antinociception. This chapter describes the efficacy and selectivity in reducing the expression of coded signaling pro- teins by in vivo administration of antisense oligodeoxynucleotides comple- mentary to their mRNA sequences. Functional data are also provided in order to assess the physiological relevance of opioid receptors and GTP- binding protein subtypes. Methods Synthesis of Oligodeoxynucleotides Synthetic end-capped phosphorothioate antisense oligodeoxynucleo- tides (ODNs) are prepared by solid-phase phosphoramidite chemistry 19 using a CODER 300 DNA synthesizer (Du Pont; Wilmington, DE) at the 1-/.~mol scale (Tables I and II). The introduction of phosphorothioate linkages is achieved by tetraethylthiuram disulfide (TETD) sulfurization. 2° 10 H. Ueda, Y. Yoshihara, H. Misawa, N. Fukushima, M. Ui, H. Takagi, and M. Satoh, J. Biol. Chem. 264, 3732 (1989). 11 S. F. Law, S. Zaina, R. Sweet, K. Yasuda, G. I. Bell, J. Stadel, and T. Reisine, Mol. PharmacoL 45, 587 (1994). 12 y. F. Lui, K. H. Jacobs, M. M. Rasenick, and P. R. Albert, J. Biol. Chem. 269, 13880 (1994). 13 S. Offermanns, T. Wieland, D. Homann, J. Sandmann, E. Bombien, K. Spicher, G. Schultz, and K. H. Jakobs, Mol. Pharmacol. 45, 890 (1994). 14 C. W. Taylor, Biochem. J. 272, 1 (1990). 15 A. D. Strosberg, Eur. J. Biochem. 196, 1 (1991). 16 j. Garz6n, M. A. Castro, J. L. Juarros, and P. S~inchez-Bl~izquez, Life Sci Pharmacol. Lett. 54, PL191 (1994). 17 j. Garz6n, A. Garcla-Espafia, and P. S~inchez-Bl~izquez, J. Pharmacol. Exp. Then. 281, 549 (1997). 18 j. Garz6n, M. Castro, and P. Sgnchez-Bl~izquez, Eur. J. Neurosci. 10, 2557 (1998). 19 M. D. Matteucci and M. H. Carouthers, J. Am. Chem. Soc. 103, 3185 (1981). 20 H. Vu and B. L. Hirschbein, Tetrahedron Lett. 32, 3005 (1991). [ 1] ANTISENSE AND OPIOID RECEPTOR SIGNALING IN CNS 5 TABLE I OLIGODEOXYNUCLEOTIDES TO Ix- AND ~-OPIOID RECEPTOR mRNA Receptora 5'-Sequence-3' Code Ref. IX (r/m) Ix (m) Ix (r/m) Ix (r/m) Ix (r/m) 6 (m) 6 (m) 6 (r) C'G* CCCCAGCCTCTTCCT* C*T b Ixurf d, e C*T*GATGTrCCCTGGG*C*C Ixl6 32 e, f T*T*GGTGGCAGTCTTCATTTT*G*G /2,291 311 d, g T*G*AGCAGGTTCTCCCAGTAC*C*A IX677-697 d~ g G*G*GCAATGGAGCAGTrTC*T*G /,1,1175_1194 d, g G*C*ACGGGCAGAGGGCACC*A*G ~7 26 f~ h, i A*G*AGGGCACCAGCTCC*A*T ~29-46 f~ J A*C*TGCAGCTCCGCA*G*G 622_37 f "The oligonucleotides correspond to those described in the code of the rat (r) or mouse (m) opioid receptor gene sequence. h An asterisk (*) indicates the phosphorothioate linkages. c This ODN is directed to a specific 5' untranslated region of the/z-opioid receptor clone. ,l G. Rossi, Y X. Pan, J. Cheng, and G. W. Pasternak, Life Sci Pharmacol. Lett. 54, PL375 (1994). e p. S~inchez-Bl~izquez, M. Rodrfguez-Dfaz, I. DeAntonio, and J. Garz6n, J. Pharmacol. Expt. Ther., in press. fP. Sfinchez-Bl~izquez, A. Garcfa-Espafia, and J. Garz6n, J. Pharmacol. Exp. Ther. 280, 1423 (1997). g G. Rossi, L. Leventhal, Y X. Pan, J. Cole, W. Su, R. J. Bodnar, and G. W. Pasternak, J. Pharmacol. Exp. Ther. 281, 109 (1997). h j. Lai, E. J. Bilsky, R. B. Rothman, and F. Porreca, Neuroreport 5~ 1049 (1994). i p. S~nchez-Blfizquez and J. Garz6n, J. Pharmacol. Exp. Ther. 285, 820 (1998). J K. M. Standifer, C C. Chien, C. Wahlestedt, G. P. Brown, and G. W. Pasternak, Neuron 12, 805 (1994). Solvents and reagents are obtained from Cruachem Ltd. (Glasgow, UK) and TETD is from Applied Biosystems (Foster City, CA). The efficiency of each base addition is higher than 98%. After synthesis the protecting groups are removed by treatment with 32% (v/v) aqueous NH3 at 55 ° overnight. Crude ODNs are purified by reversed-phase chromatography using high-performance liquid chromatography (HPLC) (Kontron, Zurich, Switzerland). Evaporated ODNs (Speed Vac Plus; Savant, Farmingdale, NY) are then dissolved in 200 /xl of 0.1 M triethylammonium acetate (pH 7.0) and injected into a C18 reversed-phase column (Spherisorb ODS-2, 5/xm; 150 × 4.6 mm) using 0.1 M triethylammonium acetate (pH 7.0) and acetonitrile as the mobile phase. The column is eluted with a 15-35% acetonitrile gradient over a 30-min period. The collected products (2 ml) are evaporated in fractions of 100/xg and stored at -20 ° until use. [...]... this antisense < /b> "knockdown" have been characterized by means of, e.g., behavioral experiments Antisense < /b> knockdown may represent a rational method by which to study the physiological and possibly pathophysiological roles played by individual GABAA receptor subunit proteins We have applied antisense < /b> technology with the purpose of selectively inhibiting the expression of GABAA receptor subunits in particular... The membranes were incubated with anti-/x- and anti-fi-opioid receptor antibodies at 1:1000 dilution in TBS-0.05% (v/v) Tween 20 (TTBS) at 6° for 24 hr After removing the antibodies the blots are washed with TTBS Secondary antiserum [goat antirabbit IgG (H + L) horseradish peroxidase conjugate (Bio-Rad)] diluted 1 : 3000 in TTBS is left for 3 hr The unbound secondary antiserum is then washed as before... decreases in the binding of radioligands to the ion channel domain and the GABA-binding site of the GABAA receptor complex 1° Therefore, the 5/2 subunit antisense < /b> ODN treatment may be viewed as a method to downregulate "benzodiazepine-site carrying" GABAA receptor complexes in particular rat brain regions Rats treated with a unilateral intrahippocampal infusion of 72 subunit antisense < /b> ODN are viable and do... particular rat brain regions in vivo We have focused on investigating the consequences of inhibited expression of one major GABAA receptor subunit, the 72 subunit A ~/subunit is essential for the presence of a high-affinity benzodiazepine binding site within a GABAA receptor complex 7 The predominant y2 subunit is a constituent of the majority of GABAA receptor complexes in the brain? We have studied biochemical,... radioligand-binding assays are often used to monitor antisense < /b> effects For control experiments we have applied mismatch ODNs in order to use ODN sequences that resemble that of the antisense < /b> ODN as closely as possible (Table I) Nucleotide specificity of GABAA receptor 3'2 subunit antisense < /b> ODN-induced effects was supported by the observation that mismatch ODNs, in which two or four central bases of the antisense.< /b> .. never induced biological effects comparable to those induced by the antisense < /b> ODN However, a mismatch ODN with six "peripheral" base shifts, retaining a central 8-mer part < /b> of the 3"2 subunit antisense < /b> ODN, induced electroencephalographic changes, although of a later onset and weaker intensity than those induced by the antisense < /b> ODN 9 It seems plausible that this ODN is able to exert some antisense < /b> action... sequence 16'2° Target gene specificity has been supported by the replication of antisense-< /b> induced changes by a second 3"2 subunit antisense < /b> ODN, complementary to an adjacent part < /b> of the 3'2 m R N A 9 (Table I) Sense ODN, an ODN antisense < /b> to the 5' untranslated region of the 3"2 subunit m R N A as well as a dopamine D2 receptor antisense < /b> 18A D Branch, Trends Biochem Sci 23, 45 (1998) 19W J Zhu, J F Wang,... Science.] 26 ANTISENSE < /b> RECEPTOR TARGETS [21 Technical Considerations Oligodeoxynucleotide Administration Oligodeoxynucleotides do not readily penetrate the mammalian bloodbrain barrierJ 3 This observation necessitates that ODNs be administered either directly into brain parenchyma or into the cerebral ventricular system When targeting a ubiquitous brain receptor such as the GABAA receptor, it may be of high... phosphorothioate-modified ODNs All sequences have been controlled for homologies to identified rodent gene sequences in the E M B L database Assessment o f Changes Induced by Antisense < /b> Oligodeoxynucleotides: Control Experiments It has not been substantially documented that antisense < /b> ODNs can be completely target gene specific TM Antisense < /b> ODNs may best be viewed as relatively specific, often offering... washed as before with TTBS Antibody binding is detected with colorimetric substrate [3,3'-diaminobenzidine (1 mg/ml), 0.02% (v/v) hydrogen peroxide, 0.04% (w/v) nickel chloride in 0.1 M Trizma base buffer, pH 7.2] or by chemiluminescent detection (ECL; Amersham) Immunoblots of SDS-solubilized membranes from mouse striatum show immunoreactive proteins at molecular masses of about 60 and 80 kDa for /x-opioid . School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom CHERYL ROBINSON-BENION (35), Department of CeU Biology, Vanderbilt University Med- ical School, Nashville,. (37), Department of Medi- cine II, University of Freiburg, D-79106 Freiburg, Germany WOLF-BERNHARD OFFENSPERGER (37), De- partment of Medicine II, University of Frei- burg, D-79106 Freiburg,. membranes were incubated with anti-/x- and anti-fi-opioid receptor antibodies at 1:1000 dilution in TBS-0.05% (v/v) Tween 20 (TTBS) at 6 ° for 24 hr. After removing the antibodies the blots