ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
`I\
`Volume 763
`
`THE IMIDAZOLINE RECEPTOR:
`PHARMACOLOGY, FUNCTIONS,
`LIGANDS, AND RELEVANCE TO
`BIOLOGY AND MEDICINE
`
`Edited by Donald J. Reis, Pascal Bousquet, and
`Angelo Parini
`
`J
`/
`
`The New York Academy of Sciences
`New York, New York
`1995
`
`Page 1 of 28
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`II
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`VrrC
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`{6 3
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`Copyright © 1995 by the New York Academy of Sciences. All rights reserved. Under the
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`pemtitted to make fair use of the material in them for teaching and research. Pennission is
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`@) The paper used in this publication meets the minimum requirements of American National
`Standard for Information Sciences - Pemwnence of Paper for Printed Library Materials,
`ANSI 239.48-1984.
`
`Library of Congress Cataloging-in-Publication Data
`
`The imidazoline receptor : pharmacology, functions, ligands, and
`relevance to biology and medicine I edited by Donald J. Reis, Pascal
`Bousquet, and Angelo Parini .
`p. em.- (Annals of the New York Academy of Sciences ; v.
`
`763)
`Includes bibliographical references and index.
`ISBN 0-89766-935-5 (cloth : alk. paper) . -ISBN 0-89766-936-3
`(paper : alk. paper)
`I. Imidazoline-Receptors-Congresses. 2. lmidazoline(cid:173)
`Derivatives-Physiological effect-Congresses. 3. Alpha
`I. Reis, Donald J.
`adrenoceptors-Congresses.
`II. Bousquet,
`Pascal.
`Ill. Parini , Angelo.
`IV . Series.
`QII.N5 vol. 763
`[RM666.I35]
`500 s-dc20
`[615'.7]
`
`95-316
`CIP
`
`PCP
`Printed in the United States of America
`ISBN 0-89766-935-5 (cloth)
`ISBN 0-89766-936-3 (paper)
`ISSN 0077-8923
`
`Page 2 of 28
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`ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
`
`Volume 763
`July 12, 1995
`THE IMIDAZOLINE RECEPTOR:
`PHARMACOLOGY, FUNCTIONS,
`LIGANDS, AND RELEVANCE TO BIOLOGY
`AND MEDICINE a
`
`Editors
`DoNALD J. REIS, PASCAL BousQUET, AND ANGELO PARINI
`
`Conference Organizer
`DoNALD J. REIS
`
`Advisory Committee
`D. J. REIS (USA), Chairman
`
`D. ATLAS (Israel)
`P. BousQUET (France)
`D. B. BYLUND (USA)
`J. P. CHALMERS (Australia)
`J. A. GARCIA-SEVILLA (Spain)
`M. GbTHERT (Germany)
`J. H. LARAGH (USA)
`
`A. P ARINI (France)
`J. L. REID (UK)
`R. R. RuFFOLO, JR. (USA)
`S. ScHAFER (Germany)
`P. M. V ANHOUTfE (France)
`J. E. WIKBERG (Sweden)
`
`CONTENTS
`
`Preface. By DoNALD J. RE1s, PASCAL BousQUET, and ANGELO
`PARINI.. ... . .... .. . . .. .... . .. .. . . . . .. .. .. .. .... . .... ...... .
`
`XV
`
`Part I. hnidazoline and Alpha-2 Adrenergic Receptors
`Pharmacological Characteristics of Alpha-2 Adrenergic Receptor
`Subtypes. By DAVID B. BYLUND............................ .
`
`Possible Structural and Functional Relationships between
`Imidazoline Receptors and a 2-Adrenoceptors. By J. PAUL
`HIEBLE and RoBERT R. RuFFoLo, JR. . . . . . . . . . . . . . . . . . . . . . . . . .
`
`1
`
`8
`
`a This volume is the result of a conference entitled II International Symposium on Imidazoline
`Receptors held in New York, New York on July 19-20, 1994.
`
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`11-Imidazoline Receptors. Definition, Characterization, Distribution,
`and Transmembrane Signaling. By PAUL ERNSBERGER,
`MARILYN E. GRAVES, LYNETIE M. GRAFF, NASSER ZAKIEH,
`PHONG NGUYEN, LAURA A. COLLINS, KELLY L. WESTBROOKS,
`and GuiNIVERE G. JoHNSON..................................
`Dynamic Properties of h-Receptors. By JARL E. S. WIKBERG.......
`Imidazoline Receptors, Subclassification, and Drug-Induced
`Regulation. By C. A. HAMILTON . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Nonadrenergic Binding of eH]Atipamezole in Rat Lung. A Novel
`Imidazole Binding Site? By BIRGIITA SJ6HOLM, JuHA-MATTI
`SA VOLA, and MIKA ScREINlN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Adrenergic and Imidazoline Receptor-Mediated Responses to UK-
`14,304-18 (Brimonidine) in Rabbits and Monkeys. A Species
`Difference. By JAMES BuRKE, ALEX KHARLAMB, TANVEER
`SHAN, EILEEN RuNDE, EowrN PADILLo, CYNTHIA MAN-LAPAZ,
`and LARRY WHEELER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`2,6-Dimethyl Clonidine: arAdrenoceptor or I1-Imidazoline
`Receptor Agonist? By P. ERNSBERGER, P. LI, and D. D. SMYTH
`
`Part II.
`
`Imidazoline Receptors: Structure, Pharmacology, and
`Regulation
`Imidazoline Binding Sites. Pharmacological and Molecular
`Characteristics. By ANGELO PARINI............. . . . . . . . . . . . . . .
`Use of High Affinity, Radioiodinated Probes for Identification of
`Imidazoline/Guanidinium Receptive Sites. By STEPHEN M.
`LANIER, BILJANA LANIER, VENKATESALU BAKTHAVACHALAM,
`CHRISTOPHER R. McGRATH, and JoHN L. NEUMEYER. .. ........
`eH]Cirazoline as a Tool for the Characterization of Imidazoline
`Sites. By l ANGEL, M. LE Rouzrc C. PIMOULE, D. GRAHAM,
`and S. ARBTLLA........... . . .. . .. .... ... ......... . ..........
`Functional Studies of Specific Imidazoline-2 Receptor Ligands. By
`D. J. Nurr, N. FRENCH, S. HANDLEY, A. HuDsoN, S.
`HusBANDS, H. JACKSON, S. JoRDAN, M. D. LALIES, J. LEWIS,
`L. LroNE, N. MALLARD and J. PRATI . . . . . . . . . . . . . . . . . . . . . . . .
`Antiidiotypic Antibodies as Tools to Study Imidazoline Receptors.
`By FATLMA .BENNAt, HUGUES GRENEY, ANNrcK MouNES,
`PAsCAL BousQUET and MoNIQUE DoNTENWrLL . . . . . . . . . . . . . . .
`Structure-Affinity Relationship of Cirazoline Derivatives for
`Idazoxan Imidazoline-Specific Sites in the Human Cerebral
`Cortex and the Rabbit Kidney . By L. BRASIL!, M. PIGINI,
`J.D. EHRHARDT, A. MoLlNES, H. GRENEY, P. BousQUET, and
`M. DONTENWILL ..... . ....... . . ... .. . ... .......... . .........
`
`22
`43
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`57
`
`66
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`78
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`96
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`100
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`106
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`112
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`125
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`140
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`149
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`Pancreatic J3-Cells Express an lmidazoline Binding Site That Is
`Distinct from 11 and 12 Sites. By SusAN L. F. CHAN, CoLIN A.
`BROWN, KAY E. ScARPELLO, and NoEL G. MoRGAN...........
`
`Imidazoline Binding Sites on PC12 Cells and Bovine Chromaffin
`Cells. By G. STEFFEN, A. DENDORFER, and P. DoMlNIAK...... .
`
`Optimization of Radioligand Binding Assays for 11-Imidazoline
`Sites. By PAUL ERNSBERGER, JoHN E. PILETZ, LYNETTE M.
`GRAFF, and MARILYN E. GRAvEs........... .... ..............
`
`Pharmacological and Immunological Characterization of Solubilized
`130-140- and 66-kD Imidazoline Receptorsjn the Rat Brain.
`By PABLO v. ESCRIBA, ANDRES 0ZAITA, ANTONIO MIRALLES,
`DoNALD J. REis, and JEsus A. GARdA-SEVILLA...............
`eH]Idazoxan Binds to Mitochondrial h lmidazoline Binding Sites
`in Isolated Cells from Rabbit Kidney Proximal Tubule. By
`C. GARGALID!s-MouDANOS and A. PARINI . . . . . . . . . . . . . . . . . . . .
`
`Castration of Rats Upregulates the Imidazoline Receptor in
`Prostate. By Y. NAssiR, S. REGUNATHAN, D. J. REis, E. D.
`VAUGHAN, JR., and D. FELSEN...............................
`
`Part III. Imidazoline Receptor Distribution
`Irlmidazoline Receptors in the Healthy and Pathologic Human
`Brain. By JESUS A. GARdA-SEviLLA ANTONIO MtRALLES,
`MAGDALENA SASTRE, PABLO V. Es RIBA, GABRrEL OLMOS, and
`J. JAVIER MEANA .... .. .... . .. .. .... . . .. ..... ........ .. .....
`
`Distribution of Nonadrenergic eH]Rilmenidine Binding in Rat
`Brain and Kidney. By P. R. KING, S. SuZUKI, W. J. LoUIS, and
`A. L. GUNDLACH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Distribution of Imidazoline Receptor Binding Protein in the Central
`Nervous System. By DAVID A. RuGGIERO, SouNDARARAJAN
`REGUNATHAN, HoNG WANG, TERESA A. MILNER, and DoNALD
`J. REIS................................... .... ..... .. ...... .
`
`In Situ Hybridization. A Complementary Method to Radioligand(cid:173)
`Mediated Autoradiography for Localizing Adrenergic, Alpha-2
`Receptor-Prpducing Cells. By ANTHONY P. NICHOLAS, VINCENT
`PIERIBONE, AKE DAGERLIND, BJoRN MEISTER, RoBERT ELDE,
`and TOMAS H6KFELT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Part IV. Signal Transduction
`Potassium Channels, Imidazolines, and Insulin-Secreting Cells. By
`MARK J. DUNNE, ELIZABETH A. HARDING, JoNATHAN H.
`JAGGAR, PAUL E. SQUIRES, RIXIN LIANG, CHARLOTTE KANE,
`RoGER F. L. JAMES, and NicHOLAS J. M. LoNDON. . . . . . . . . . . . .
`
`153
`
`157
`
`163
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`169
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`172
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`174
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`178
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`194
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`208
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`222
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`243
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`A New Therapeutic Approach for Sickle Cell Disease. Blockade of
`the Red Cell Ca2•-Activated K+ Channel by ClotrimazoJe. By
`CARLO BRUONARA, LuerA DE FRANCESCHI, CARRIE C. ARMSBY,
`NACERA SAADANE, MARIE ThuoaL, Yves BEUZARD, ANN
`RLITENHOUSE, NADER RlFAI, ORAI-I PLAir, and SETH L. ALPER .
`
`Clonidine and Cirazoline Inhibit Activation of Nicotinic Channels
`in PC-12 Cells. By I. F. MusGRAVE, D. KRAuTWURST,
`J. HEscHELER, and G. ScHULTZ..............................
`
`CaJcium Channel-Dependent and 11-Imidazoline Receptor Binding
`Properties of 2-(4'-lsothiocyanatobenzyl) Imidazoline Analogs
`in Vascular and Brain Tissues. By L. LEI, M. SLAVICA,
`P. ERNSBERGER, M. E. GRAVES, P. PATIL, D. MILLER. and
`D. FELLER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Inhlbition of Red Cell Ca2•-Activated K + Transport by Clotrimazole
`Does Not Take Place via lmidazoline Bincling Sites. By
`I. COUPRY, C. C. ARMSBY, S. ALPER, A. PARINL, and C.
`BRUONARA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Effects of Rilmenidine on Signal Transduction Mechanisms
`Associated with a 2-Adrenergic and Imidazoline Receptors in
`Brain. By SouNDARARAJAN REouNATHAN, SoNJA BRAMWELL,
`and DoNALD J . . REis.............. ... ..................... . .
`
`Part V. Endogenous Ligands
`Endogenous Ligands of lmidazoline Receptors: Classic and
`Immunoreactive Clonidine-Displacing Substance and
`Agmatine. By DoNALD J. RE1s, GsN L1, and SooNDARARA.IAN
`R.EOUNATHAN.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Molecular and Physiological Properties of Clonidine-Displacing
`Substance. By DAPHNE ATLAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Agmatine Is Synthesized by a Mitochondrial Arginine
`Decarboxylase in Rat Brain. By GEN L1, SouNDARARAJAN
`REOUNATHAN, and DONALD J. REIS. . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Agmatine Is Widely and Unequally Distributed in Rat Organs. By
`WALTER RAASCH, SouNDARARA.IAN REouNATHAN, GEN L1, and
`DONALD J. REIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`(mmunopurification of a Vasopressive Substance from Human
`Serum with an Anti-Clonidine Antibody. By M. DoNTENWrLL,
`A. MOL£NES, F. BENNAI, J. FELDMAN, G. BRICCA,
`A. BELCOURT, and P. BOUSQUET . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`262
`
`272
`
`283
`
`287
`
`290
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`295
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`314
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`325
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`330
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`335
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`Part VI. Cellular Actions: Kidney, Pancreas, Adipocytes, Trachea
`Imidazoline Receptors and Sodium Excretion in the Rat Kidney. By
`DoNALD D. SMYTH, FRANCIS K. DARKWA, and S. BRIAN
`PENNER....................................................
`
`340
`
`Rilmenidine Alters Renal Function When Administered
`Intracerebroventricularly or Intrarenally. By S. BRIAN PENNER
`and DoNALD D. SMYTH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`a 2 Adrenoceptor- and Imidazoline-Preferring Binding Sites in the
`Dog Kidney. By RoGER G. EVANS and JoHN M. HAYNES . . . . . .
`
`Characterization of the Imidazoline Binding Site Involved in
`Regulation of Insulin Secretion. By NoEL G. MoRGAN, SusAN
`L. F. CHAN, CoLIN A. BRowN, and EFrHYMIA TsoLI...........
`
`Imidazolines Stimulate Release of Insulin from RIN-5AH Cells
`Independently from 11- and lz-lmidazoline Receptors. By
`GABRIEL OLMOS, RoHIT N. KuLKARNI, MuNIRUL HAQUE, and
`JoHN MAcDERMOT..........................................
`
`Influence of Clonidine on Alloxan-Induced Diabetes in Rats. By
`MIHAl NECHIFOR, CRISTIANA FILIP, ELENA TESLARIU, BoGDAN
`I. NEUGHEBAUER, MARCEL CosTULEANU, and ADRIANA NEGRU..
`
`Nonadrenergic Imidazoline Binding Sites and Amine Oxidase
`Activities in Fat Cells. By CHRISTIAN CARPENE, Luc MARTI,
`ALAN HuDsoN, and MAX LAFONTAN ........................ .
`
`Evidence for a Role for Imidazoline I 1 Binding Site in Rat Brown
`Adipocytes. By G. MoRY, F. TEssoN, M. CoMBES-GEORGE,
`M. NECHAD, and A. PARINI..................................
`
`Regulation of Electrolyte Transport in Rabbit Tracheal Epithelial
`Cells by the I 1- Imidazoline Agonist Moxonidine. By CAROLE
`M. LIEDTKE and PAUL ERNSBERGER..........................
`
`353
`
`357
`
`361
`
`374
`
`377
`
`380
`
`398
`
`401
`
`Part VII. Cellular Actions: CNS and Autonomic Nervous System
`az-Adrenoceptor-Independent Inhibition by Imidazolines and
`Guanidines of Noradrenaline Release from Peripheral, but Not
`Central Noradrenergic Neurons. By M. G6THERT, G. J.
`MoLDERINGS, K. FINK, and E. ScHLICKER. . . . . . . . . . . . . . . . . . . . .
`
`405
`
`No Relationship of 11- and Iz-Imidazoline Binding Sites to
`Inhibitory Effects of Imidazolines on Ligand-Gated Ion
`Channels. An Investigation in the Adrenal Medulla and in
`Neuroblastoma Cells. By G. J. MoLDERINGS, K. RuPPERT,
`H. B6NISCH, and M. G6THERT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Moxonidine Decreases Gastric Secretion and Gastric Mucosal
`Injury in Rats. By G. B. GLAVIN and D. D. SMYTH............
`
`420
`
`433
`
`J
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`Role of the Epithelium in the Release of Contractile Agents from
`the Rat Vas Deferens by Clonidine. By AFoNso CARJCATI(cid:173)
`NETO, ANDRES. PuPO, Al.MJR G. WANDERLEY, MANUEL
`NUNEZ-VF!RGARA, IvAN Ho JuN KoH, NmnE H. JURKIEWicz,
`and ARON JURKLEWlCZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Coexpression of lmidazoline Receptors and Agmatine in Rat
`Carotid Body. By CHARLOTTE YoUNGSON, SouNDARARAJAN
`REouNATHAN, HoNG WANG, GEN Lr, and DoNALD J. RErs. . . . . .
`
`Effect of 11-lmidazo]jne Receptor Activation on Responses of
`Hypoglossal and Phrenic Nerve to Chemical Stimulation. By
`M. A. H AXHTU, I. A. DRBSHAJ, B. EROKwu, L. A. CoLLms, and
`P. ERNSBERGER. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Centrally Mediated Ocular Hypotension: Potential Role of
`lmidazoline Receptors. By W. RICHARD CAMPBELL and DAVID
`E. P011ER..................................................
`Chronic Imidazoline Drug Treatment Increases the
`Immunoreactivity of Glial Fibrillary Acidic Protein in Rat
`Brain. LSL 60101 as a Novel and Selective Ligand for 12 -
`Imidazoline Receptors. By JESUS A. GARciA-SEvrLLA, REGINA
`ALEMANY, GABRIEL OLMos, ANGEL MENARGUl!s, and RosBND
`0BACI-l. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`In Vivo Modulation of Norepinephrine and Glutamate Release
`through lmidazoljne Receptors in the Rat Central Nervous
`System. By J. J. MEANA, M. HERRERA-MARSCHrrz, and
`M. GorNY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Effects of the Ir lmidazoline Receptor Ligand LSL 6010 1 on
`Various Models of Anorexia in Rats. By ANOBL MENARGUES,
`MAR CED6, OLGA ARnoA, RossEND 0BACH, and JEsus A.
`GARCiA-SEVILLA............................................
`
`Spinal Imidazoline Receptors Do Not Mediate the Antinociceptive
`Action of Intrathecal Clonidine in the Rat. By PHILIP J.
`MONROE, 0BBORAH L. SMITH, and DAvro J. SMITH .... .... . ...
`
`The Stimulatory Effect of Clonidine on Locus Coeruleus
`Noradrenergic Neurons through Imidazo]jne Receptors Is
`Modulated by Excitatory Amino Acids. By JOSEBA PtNEDA,
`1. ANGEL RUiz-ORTEGA, LUISA UGEDO, and Jesus A.
`GARdA-SEVILLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Chronic Treatment with Phenelzine and Other Irreversible
`Monoamine Oxidase Inhibitors Downregulates 12-Imidazo]jne
`Receptors in the Brain and Liver. By REGINA Al.BMANY,
`G ABRIEL OLMos, and JESus A. G ARciA- SEVILLA . • . . . . . . . . . . . . .
`
`436
`
`440
`
`445
`
`463
`
`486
`
`490
`
`494
`
`497
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`501
`
`506
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`Involvement of I1-Imidazoline Receptors in Mood Disorders. By
`JoHN E. PrLETZ and ANGELOS HALARIS . . . . . . . . . . . . . . . . . . . . . . .
`
`510
`
`Decreased Number and Immunoreactivity of Iz-Imidazoline
`Receptors in the Frontal Cortex of Suicide Victims. By
`MAGDALENA SASTRE, PABLO V. EscRIBA, DoNALD J. RErs, and
`JESUS A. GARdA-SEVILLA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Human Cerebral Imidazoline Binding Sites. Further Biochemical
`Investigations. By H. GRENEY, M. DoNTENWILL, G. BRrccA,
`A. BELCOURT, and P. BousQUET . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Part VIII. Cardiovascular System and Hypertension
`Imidazoline Receptors and Cardiovascular Regulations. A
`Statement. By PASCAL BouSQUET, HuGUES GRENEY, FATIMA
`BENNAI, JosrANE FELDMAN, JEANNE STUTZMANN, ALAIN
`BELCOURT, and MoNIQUE DONTENWILL . . . . . . . . . . . . . . . . . . . . . . .
`
`Importance of Imidazoline Receptors in the Cardiovascular Actions
`of Centrally Acting Antihypertensive Agents. By GEOFFREY
`A. HEAD.................................................. .
`
`Alpha-2 Adrenergic Receptors. Immunohistochemical Localization
`and Role in Mediating Inhibition of Adrenergic RVLM
`Presympathetic Neurons by Catecholamines and Clonidine. By
`R. L. STORNETTA, D. HUANGFU, D. L. ROSIN, K. R. LYNCH,
`and P. G. GuYENET........................................ .
`
`Mechanism of Sympathoinhibition by Imidazolines. By BELA SzABO
`and RoLF URBAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`a-Adrenoceptors and GABA Receptors in the Hypertensive and
`Hypotensive Actions of Noradrenaline Intrathecally Injected in
`Anesthetized Rats. By M. C. GARCiA and S. M. CELUCH. . . . . . .
`
`Imidazoline and Adrenoceptor Involvement in Cardiovascular
`Responses to Clonidine in Sham and Sinoaortic Denervated
`Rats. By D. RICCI and C. A. TAIRA . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Influence of Imidazolines on Catecholamine Release in Pithed
`Spontaneously Hypertensive Rats. By W. HAUSER, J. GUTTING,
`TH. NGUYEN, and P. DoMINIAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Imidazoline Receptors in Vascular Smooth Muscle and Endothelial
`Cells. By SouNDARARAJAN REGUNATHAN, CHARLOTTE
`YouNGSON, HoNG WANG, and DoNALD J. REis............... .
`
`520
`
`523
`
`526
`
`531
`
`541
`
`552
`
`566
`
`570
`
`573
`
`580
`
`Page 9 of 28
`
`SLAYBACK EXHIBIT 1028
`
`

`

`The Relationship between the Adrenoceptor and Nonadrenoceptor(cid:173)
`Mediated Effects of Imidazoline- and Imidazo le-Containing
`Compounds. By J. C. Mc GRATH, C. M. BROWN, C. J. DALY,
`D. KENDALL, A. MACKINNON, D. J. MILLER, M. NAGADEH,
`A. O'DowD, J. J. O'DowD, D. PINTHONG, G. SINGH,
`A. G. B. TEMPLETON, and V. G. WILSON . . . . . . . . . . . . . . . . . . . . .
`Effect of the Imidazoline Receptor Ligand Moxonidine on Human
`Platelet Aggregation. By SALLY A. WIEST and MITCHELL
`l. STEINBERG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Role of Imidazoline Receptors in Halothane-Epinephrine
`Arrhythmias. By YuKIO HAYASHI, TAKAHIKO KAMIBAYASHI,
`ATSUSHI YAMATODANI, MASAK AZU KuRo, and IKUTO YosHIYA .
`Antiarrhythmic Effect of the Selective 11-Imidazoline Receptor
`Modulator Moxonidine on Ouabain-Induced Cardiac
`Arrhythmia in Guinea Pigs. By H.-J. MEsT, P. THOMSEN, and
`A. RAAP. .. ..... .. ............ . .... ... ..... . ..... .. ........
`Rilmenidine and the High Risk Hypertensive Patient. By JoHN H.
`LARAGH... . .... ... .... ... .... .. ... .. ..... . .................
`11 Agents: A New Approach to the Treatment of Hypertension. By
`M. DuBAR and G. PILLION .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Why Imidazo line Receptor Modulator in the Treatment of
`Hypertension? By S. G. Sc HAFER, E. C. KAAN, M. 0.
`CHRISTEN, A. Lbw- KROGER. H.-J. MEST, and 0.-J.
`MoLDERINGs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Clinical Pharmacology of Drugs Acting on Imidazotine and
`Adrenergic Receptors. Studies with Clonidine, Moxonidine,
`Rilmenidine, and Atenolol. By JoHN L. REID, VsEVOLOD
`PANFILOV , GRAHAM MAc PHEE, and HENRY L. ELLIOTT . ...... . .
`Central Nervous System Norepinephrine Turnover in Essential
`Hypertension. By GAVIN W. LAMBERT, CLAUDIA FERRIER,
`DAVID M. KAYE, GARRY L. JENNINGS, VICTOR KALFF, MICHAEL
`J. KELLY, HELEN S. Cox, ANDREA G. TuRNER, and MuRRAY D.
`ESLER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`591
`
`606
`
`610
`
`620
`
`634
`
`642
`
`659
`
`673
`
`679
`
`*****
`
`Subject Index . .. .... .. ......... . . .
`Index of Contributors . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . .
`
`695
`705
`
`Page 10 of 28
`
`SLAYBACK EXHIBIT 1028
`
`

`

`Adrenergic and lmidazoline
`Receptor-Mediated Responses to
`UK-14,304-18 (Brimonidine) in
`Rabbits and Monkeys
`A Species Difference
`
`JAMES BURKE,a ALEX KHARLAMB,
`TANVEER SHAN, EILEEN RUNDE, EDWIN PADILLO,
`CYNTHIA MANLAPAZ, AND LARRY WHEELER
`Department of Biological Sciences
`Allergan Inc.
`2525 Dupont Dr.
`Irvine, California 92715
`
`Glaucoma is a heterogenous group of ocular disorders in which there is progressive
`damage to the optic nerve, resulting in visual field loss and eventual blindncss. 1
`Elevated intraocular pressure (lOP) is an important risk factor for glaucoma? Current
`treatment of primary open angle glaucoma, tJ1e mo t common fonn, is directed
`towards the reduction of lOP. Alpha-adrenergic recertor agonist reduce lOP3 and
`have played a significant role in managing glaucoma. Brimonjdine (UK-14,304-18,
`
`AGN 190342), a relatively selective and potent alphn-2 adrenoceptor agonist,5·6 Jowers
`lOP in nom1otensive rabbits/--9 cats,8 and monkeysa· 10·11 as well as glaucomatous or
`ocular hypertensi ve rabbits,8 monkeys. 10·12 and humans. 12- 14 Brimonidine is also
`highly effective in preventing elevated lOP following ocular laser surgery. 1 17 ln
`addition to activating alfsba-2 adrenoceptors, brimonidine binds to nonadrenergic
`" imidazoline" receptors 8•19 that can mediate biological effects such as hypotension20
`and potentially TOP. This study evaluates ilie pharmacology of the ocular and cardio(cid:173)
`vascular Iesponses to topical administration of brimonidine in rabbits and monkeys
`by pretreatment wilb antagonists selective for alpha-adrenergic and imidazoline recep(cid:173)
`tors and by radioligand binding displacement experin1ents using eHlbrimonidine in
`membranes from rabbit ciliary body and monkey brain.
`
`MATERIALS AND METHODS
`
`Materials. The following compounds were used in these experiments: Brimonidine
`tartrate (UK-14,304-18), a gift from Pfizer, UK; rauwolscine HCI, prazosin HCI, and
`
`a Correspondence and requests for reprints: James Burke, Department of Biological Sciences,
`Allergan Inc., 2525 Dupont Dr., Irvine, California 92715.
`78
`
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`

`BURKE et al.: BRIMONIDINE
`
`79
`
`idazoxan HCl, purchased from Research .Biochemical Inc., Natick, Massachusetts;
`and SKF 105854, a gift from SmithKline Beecham Labs., Philadelphia, Pennsylvania
`(fomlerly SmithKiine Beckman). Drug concentration in the in vivo experiments are
`expressed as a percentage (w/v) of the active ingredient, that is, base. The radioligand
`eHJbrimonidine (UK-14,304; specific activity 63 Cilmmol) was obtained from New
`England Nuclear, Boston, Massachusetts.
`In Vivo. Experiments were performed in fully conscious drug-naive New Zealand
`White rabbits (albino) of either sex weighing 2-3 kg and female cynomolgus monkeys
`weighing 3-4 kg. Monkeys sat in specially designed chairs (Primate Products, San
`Francisco, California) throughout each experiment and occa ionally were fed orange
`juice and fruit. Rabbits were put back into cages after each measurement. A 30R
`model Digilab pneumatonometer and a millimeter cale ruler were used to measure
`lOP and pupil diameter (PD), respectively. Pupil diameter was measured to the nearest
`0.5 mm. Blood pressure and heart rate wer measured noninvasively with a model
`BPlOOS automated sphygmomanometer from Nippon Colin, Japan. The pressure cuff
`of the sphygmomanometer was placed on the forearm of monkeys and the hindleg
`o( rabbits. Drugs were formulated in distilled water and administered unilaterally a
`a single 50-,.LI eyedrop; the contralateral eyes received an equal volume of saline
`solution. Antagonists were administered 30 minutes prior to brimonidine instillation.
`Two baseline measurements were made before instillation of brimonidine, followed
`by periodic measurements up to 6 hours postinstillation.
`
`Receptor Binding Assays
`
`Tissue Preparation. Plasma membrane homogenate was prepared from rabbit
`iris/ciliary body, monkey cerebral cortex and brain stem, and rat cerebral cortex (a
`a control). Briefly, tissues (I g) were homogenized in 25 ml of ice-cold buffer (5
`mM Tris-H..CJ, pH 7.5) using a Polytron homogenizer for 30 econds at setting #7
`and centrifuged for 10- 12 minutes at 300 x g at 4 ·c. The supernatant was filtered
`through two layers of gauze and diluted I :2 with 50 mM Tris-HCI buffer, pH 7.4,
`then centrifuged at 49,000 x g for 15 -20 minute . The pellet fraction wa washed
`three times, resu pended in Tris-HCI buffer, and centrifuged for 15- 20 minutes at
`49,000 x g. The pellet was then stored at -so•c until the binding assay.
`Binding Studies. Frozen membrane pellet was resuspended in buffer containing
`50 mM Tris-HCI, 0. L mMMnC12, at pH 7.7. Rabbit iris/ciliary body was resuspended
`in a different buffer of the following composition: 50 mM Tris-HCI, I mM MgC12,
`and 2 mM EGTA, pH 7 .5. Membrane protein homogenate (75 -300 fl.&) was incubated
`with radioligand for 90 minutes at 22•c in a final volume of 250 or 500 111. At the
`end of the incubation period, samples were filtered through glass fiber filters (What(cid:173)
`man GP/.8) in a 24- or 96-well cell harvester and rapidly washed four times witb 4
`ml of iced-cold 50 rnM Tris-liCI buJfer, pH 7.4. The filters were th.en oven dried
`and transferred to scintillation vials containing 10 ml of Beckman's Ready Protein®
`scintillation cocktail for counting. Specific binding, defined by I 0 11M phentolamine
`for competition studies, was as follows: 62% in rabbit iris/ciliary body using 2.4 nM
`eH]brimonidine and 90% in monkey and rat cerebral cortex and monkey brain stem
`using 1 nM eH]brimorudine. Protein concentrations were determined with a protein
`
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`

`

`80
`
`ANNALS NEW YORK ACADEMY OF SCIENCES
`
`assay kit from Bio Rad (Hercules, Califomia). Binding i
`therms and equilibrium
`dissociation and affiJlity constants were analyzed and determined by the nonlinear
`least squares curve fitting program AccuFit Competition/Saturation from Beckman,
`Fullenon, California.
`Data Presentation. Data from in viv experiments are presented in graph fonn
`as the mean percentage of change from pre-drug baseline or absolute mean :!: standard
`error ver us time or in table form as mean percentage of change from ba cline.
`Statistica.l ana lysis of the response to brimonidine or the antagonists was done by a
`paired Student's t test comparing data at each time point with pretreatment values
`(baseline) except for the rabbit lOP data. intraocular pressure in rabbits tended to
`increa e with time due to diurnal influences, so lOPs were compared between the
`drug-treated group and a separate vehicle-treated group and analyzed using an un(cid:173)
`paired Student' s t test. A p value of <0.05 was considered significant. To statistica.lly
`evaluate the effect of antagonist pretreatment on the response to brimonidine, mean
`percentages of change from pre-drug levels in each experimental group were computed
`and compared with the mean percentage of change from the brimonidine only group
`at each time point us ing the unpaired Student's r test; p < 0.05 wa~ considered
`significant.
`
`RESULTS
`
`Rabbits
`
`The lOP response to multiple concentrations 0.000 I -0.1% of unilateral ocular
`administration of brimonidine were determined in rabbits (Fro. l). Starting lOPs
`ranged from 18-22 rom Hg. Brimonidine produced a concentration-dependent de(cid:173)
`crease in IOP. The 0.01% concentration was at the top of the concentration-response
`curve and produced a peak ocular hypotensive re ponse of 23 :!: 2% at 2 hours after
`drug administration. Higher concentrations (0.03 and O.J %) increased the duration
`of action and produced a contralateral decrease in JOP. The 0.1% concentration
`produced ocular hypotension in the treated eye whicl1 lasted the duration of the 6-
`hour experiment. TheJOP decrease in eyes treated with 0.0 l , 0.03 and O.J lJl brimoni(cid:173)
`dine wa preceded by transient oculnr hypenension. There were no pupillary responses
`to brimonidine in rabbits at the concentrations tested.
`The effects of ocular administration of 0.03, 0.1, and 0.3% brimonidine on blood
`pressure and heart rate in rabbits are illustrated in FIGURE 2. Pre-drug blood pressure
`was about I 00150 mm Hg. Heart rate was about 230 beats/min. Brimonidine (0.03,
`0.1%) did not produce a stati ·tically significant change in blood pressure or heart
`rate. The 0.1% concentration reduced systolic blood pressure by 12 :!: 8%. Systolic
`ignificantly reduced with the 0.3% concentration by 22 :!: 6%
`blood pressure was
`at 1 hour following instillation of brimonidine. Heart rate was suppressed by 15 ±
`391 at I hour. The hypotensive and bradycardic effects of brimonidine in rabbits
`were resolved by 2 hours after drug administration.
`The effects of topical antagonist pretreatment on the lOP response to 0.01 and
`0.03 % brimonidine in rabbits are presented in TABLE 2. The antagonists rauwolscine,
`idazoxan, or SKF I 05854 at the I% concentration did not significantly alter TOP
`
`Page 13 of 28
`
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`

`

`BURKE et al.: BRIMONIDINE
`
`81
`
`26
`
`VEHICLE ( 16)
`
`28
`
`0.0001% BRIMONIDINE (9)
`
`a; 2.2
`:I:
`E 20
`5
`0..
`Q 18
`
`26
`
`22
`
`20
`
`18
`
`24 ~ 24
`
`. ·H
`
`16
`
`14
`· 1
`
`28
`
`t
`
`0
`
`1
`
`2
`
`3
`
`4
`
`0.01% BRIMONIDINE (25)
`
`16
`
`14
`
`12
`-1
`
`26
`
`*
`t*
`
`0
`
`--+--TREATED
`·· · 0
`·FELLOW
`
`3
`
`4
`
`5
`
`I
`6
`
`0.03% BRIMONIDINE (37)
`
`28
`
`26·
`
`0.001% BRIMONIDINE (9)
`
`24 ~
`
`.
`
`* *
`
`t
`
`---- TREATED
`0 FELLOW
`
`0
`
`3
`
`4
`
`5
`
`22
`
`20
`
`18
`
`16
`
`14
`
`12
`-1
`
`28
`
`26
`
`24
`a; 22
`:I:
`E 20
`5
`0.. 18
`Q
`
`_.¢ · ~ -
`
`*
`
`16
`
`14
`
`12
`· 1
`
`t
`
`0
`
`*
`
`---TREATED
`- 0 FELLOW
`
`3
`2
`1
`TIME (HRS. )
`
`4
`
`5
`
`6
`
`24
`
`18
`
`16
`
`14
`-1
`
`t
`
`0
`
`*
`
`0
`
`·FELLOW
`
`2
`1
`3
`TIME (HRS.)
`
`4
`
`5
`
`6
`
`26
`
`24
`
`22
`
`20
`
`18
`
`16
`
`14
`
`12
`-1
`
`0.1% BRIMONIDINE (21)
`*
`
`¢ -
`
`/1 '
`
`-0
`
`*
`
`*
`__._TREATED
`0 FELLOW
`
`*
`
`~
`
`t t ~ . . 1
`
`•'
`
`0
`
`2
`1
`3
`TIME (HRS.)
`
`4
`
`5
`
`FIGURE 1. Intraocular pressure responses to unilateral topical administration of saline solution
`and 0.000 I -0.1% brimonidine in conscious albino rabbits. Saline or brimonidine was adminis(cid:173)
`tered as a single 50-f1l drop. The treated eye is indicated by -•-. the untreated eye (fellow)
`by -0-. Values in parentheses represent the number of rabbits in each experiment. Arrows
`indicate time of drug administration. Asterisks indicate a statistically significant (p < 0.05)
`deviation from vehicle control for the treated eye; daggers indicate a statistically significant
`(p < 0.05) deviation from vehicle control for the untreated eye.
`
`during the ocular hypotensive phase of brimonidine. Prazosin, 0.3%, lowered lOP
`by about 13%. The 0.01 and 0.03% concentrations of brimonidine decreased lOP by
`23 and 20%, respectively. Rauwolscine inhibited the ocular hypotensive response to
`brimonidine by about 70%. Idazoxan inhibited the brimonidine-induced lOP decrease
`by about 37%. Neither SKF 105854 nor prazosin inhibited the lOP response to
`brimonidine in rabbits .
`
`Monkeys
`
`The effects of brimonidine (