Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1058 _____________________________ _____________________________ α1- and α2-Adrenoceptors in the Eye Pharmacological and Functional Characterization BY ANNA WIKBERG MATSSON ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2001 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Neuroscience presented at Uppsala University in 2001 ABSTRACT Wikberg-Matsson A. 2001. α1- and α2-Adrenoceptors in the Eye. Pharmacological and Functional Characterization. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1058. 66 pp. Uppsala. ISBN 91-554-5077-6. α1- and α2-Adrenoceptors are involved in various physiological events in the eye: blood flow regulation, aqueous humor dynamics and pupil regulation. The α1- and α2- adrenoceptors can be further subdivided into six subtypes (α1A, α1B, α1D, α2A ,α2B, and α2C ). Currently available α1- and α2-adrenergic drugs are not selective for the different subtypes and some ocular adrenergics have undesirable side-effects, both local and systemic. A better understanding of the subtype distribution in the eye would be useful when designing new drugs with greater efficacy and fewer adverse effects; this applies especially to the treatment of glaucoma. The purpose of the thesis was therefore to identify and localize the different subtypes of α1- and α2- adrenoceptors in the eye. The identities of the α1-adrenoceptor subtypes were studied in various parts of pig and albino rabbit eyes by radioligand binding. In the pig retina and in the albino rabbit iris, ciliary body and retina, mixed populations of α1A- and α1B-adrenoceptors were localized. In the rabbit choroid only the α1A-adrenoceptor subtype was detected. The α2-adrenoceptor subtypes were also characterized by radioligand binding, in different parts of the pig eye. In the iris, ciliary body and choroid, only α2A- adrenoceptors were localized, while in the retina, mostly α2A-adrenoceptors and a minor population of α2C-adrenoceptors were identified. High densities of α2A- adrenoceptors were found in the ciliary body and choroid. The effect of α2-adrenoceptor agonists on the porcine ciliary artery was studied on a small-vessel myograph. α2-Adrenoceptor agonists proved to be potent vasoconstrictors in the porcine ciliary artery and it was found that the vasoconstriction induced by brimonidine was mediated by the αA-adrenoceptor. Keywords: α1-adrenoceptor subtypes, α2-adrenoceptor subtypes, iris, ciliary body, choroid, retina, ciliary artery Anna Wikberg-Matsson, Department of Neuroscience, Ophthalmology, Uppsala University Hospital, S-751 85 Uppsala, Sweden Anna Wikberg-Matsson ISSN 0282-7476 ISBN 91-554-5077-6 Printed in Sweden by Reprocentralen, Ekonomikum, Uppsala 2001 To my family The thesis is based on the following papers which will be referred with to by roman numerals (I-V): I. Wikberg-Matsson A, Wikberg JES, Uhlén S. (1995). Identification of α α α drugs subtype-selective for 2A-, 2B- and 2C-adrenoceptors in the pig cerebellum and kidney cortex. Eur. J. Pharmacol. 284:271-279. II. Wikberg-Matsson A, Wikberg JES, Uhlén S. (1996). Characterization α α of 2-adrenoceptor subtypes in the porcine eye: Identification of 2A- α α adrenoceptors in the choroid, ciliary body and iris, and 2A-and 2C- adrenoceptors in the retina. Exp. Eye Res. 63:57-66. III. Wikberg-Matsson A, Wikberg JES, Uhlén S. (1998). Characterization of α1-adrenoceptor subtypes in the pig. Eur. J. Pharmacol. 347:301-309. IV. Wikberg-Matsson A, Uhlén S, Wikberg JES. (2000). Characterization of α 1-adrenoceptor subtypes in the eye. Exp. Eye Res. 70:51-60. α V. Wikberg-Matsson A, Simonsen U. (2001). Potent 2A-adrenoceptor- mediated vasoconstriction by brimonidine in the porcine ciliary arteries. (Invest Ophthalmol Vis Sci 2001) Reprints were made with permission from the publishers. CONTENTS ABBREVIATIONS 7 INTRODUCTION 9 History of adrenoceptors 9 G-protein-coupled receptors 10 Classification of α1-adrenoceptors 12 Signalling mechanisms of the α1-adrenoceptors 14 Physiological responses mediated by α1-adrenoceptors 14 Classification of α2-adrenoceptors 16 Signalling mechanisms of the α2-adrenoceptors 17 Physiological responses mediated by α2-adrenoceptors 17 Vascular α2-adrenoceptors 20 Ocular α-adrenoceptors 22 Glaucoma 23 Adrenergics in glaucoma treatment 24 AIMS OF THE PRESENT STUDY 27 MATERIALS AND METHODS 29 Animals 29 Tissue preparations for binding studies 29 In vitro studies on ciliary arteries (V) 30 Analysis of binding data (I–IV) 32 Analysis of the dose – response studies (V) 33 Drugs and chemicals 34 Statistical analysis 35 RESULTS 36 Identification of α2-adrenoceptor selective drugs in pig (I) 36 α2-adrenoceptors in pig eye (II) 36 α1-adrenoceptor subtypes in the pig and rabbit eyes (III, IV) 37 Effect of α2-adrenoceptor agonists in the ciliary artery (V) 40 DISCUSSION 41 α1-adrenoceptors in the eye 41 α2-adrenoceptors in the eye 42 CONCLUSIONS 48 IMPLICATIONS FOR FUTURE RESEARCH 49 ACKNOWLEDGEMENTS 51 REFERENCES 53 ABBREVIATIONS AA = arachidonic acid AR = adrenoceptor ANOVA = analysis of variance cAMP = cyclic 3´,5´-adenosine monophosphate ARC239 = (2-(2,4-(O -methoxyphenyl)-piperazin-1-yl)-ethyl-4,4- dimethyl-1,3(2H,4H)-isoquinolindione bFGF = basic fibroblast growth factor BMY7378 = (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8- azaspiro[4.5]decane-7,9-dione) dihydrochloride BRL41992 = (1,2-dimethyl-2,3,9,13b-tetrahydro-1H- dibenzo[c,f]imidazol-[1,5-a]azepine) BRL44408 = (2-[2H-(1-methyl-1,3-dihydroisonidole)methyl]-4,5- dihydroimidazole) CEC = chloroethylclonidine EC50 = concentration of drug producing 50% of maximum effect EDTA = ethylendiaminetetraacetic acid DAG = diacylglycerol GDP = guanosinediphosphate GPRC = G-protein-coupled receptor GTP = guanosinetriphosphate IUPHAR = International Union of Pharmacology Committee on Receptor Nomenclature IP3 = inositol 1,4,5-triphosphate Kd = dissociation constant MK912 = (2S,12bS)-1´3´-dimethylspiro (1,3,4,5´,6,6´,7,12b- octahydro-2H-benzo[b]furo[2,3-a]quinazoline)-2,4´- pyrimidin-2´-one mRNA = messenger RNA 7 pA2 = negative logarithm to base 10 of the molar concentration of an antagonist that makes it necessary to double the concentration of an agonist needed to elicit the original submaximal response PIP2 = phosphatidylinositol 4,5-biphosphate PK = protein kinase pKB = negative logarithm to base 10 of the equilibrium dissociation constant for the antagonist calculated from Schild´s analysis pKi = negative logarithm to base 10 of the dissociation constant in competition binding analysis PCR = polymerase chain reaction PLA = phospholipase A PLC = phospholipase C PSS = physiological salt solution IOP = intraocular pressure RPE = retinal pigment epithelium RX821002 = (1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline SKF104856 = 2-vinyl-7-chloro-3,4,5,6-tetrahydro-4- methylthienol[4,3,2ef[3]benzazepine Tris = tris-(hydroxymethyl)aminomethane VDCC = voltage-dependent calcium channels WB4101 = 2-(2,6-Dimethoxyphenoxymethyl)aminomethyl-1,4- benzodioxan hydrochloride 8 INTRODUCTION History of adrenoceptors Adrenoceptors are cell membrane receptors, belonging to the G-protein coupled family of receptors. The cathecholamines noradrenaline and adrenaline are the physiological agonists of the adrenoceptors. Adrenoceptors are found in nearly all peripheral tissues and in many locations in the central nervous system. The history of the adrenoceptors starts more than a century ago in 1895 with the discovery by Oliver and Schäfer of the vasopressor effect of extracts from the suprarenal gland [1]. In 1904 Stoltz succeeded in synthesizing adrenaline [2]. The existence of an adrenergic receptor was suggested by Langley, who postulated that adrenaline and other pharmacologically active substances exert their effects by interacting with ”receptive substances” [3]. The discovery of adrenaline also led to the idea that sympathetic transmission might [4] be mediated by an adrenaline-like substance . The concept of distinct adrenoceptors was developed in 1948 when Ahlqvist described two types of adrenoceptors based on the rank order of potency of a series of catecholamines [5]. The receptor designated β have a mainly inhibitory function, while α receptors are mainly excitatory. Later, also based on functional pharmacologcal evidence, the β-adrenoceptors were [6] subdivided into β1 and β2 . The next major development in adrenoceptor classification occurred in 1974, with the proposal that α-adrenoceptors could [7] be subclassified into α1-postjunctional and α2-prejunctional adrenoceptors . Subsequentely, as evidence of postjunctionally located α2-adrenoceptors accumulated, this purely anatomical classification was redefined into a pharmacological subclassification not dependent on location [8]. Further additions to our understanding of the α-adrenoceptors have derived from new pharmacological and molecular biological methodology. With the identification of potent and highly selective α1- and α2-adrenoceptor agonists and antagonists, the subdivision has come to rely on a 9 pharmacological subclassification rather than an anatomical or functional subdivision. With the advent of the radioligand binding assay in the mid-1980s, [9, 10] it was demonstrated that there are subtypes of both α1-adrenoceptors and [11, 12] α2-adrenoceptors . Further characterization has been made by applying molecular biology technology. Six genes for α-adrenoceptors have now been identified and sequenced (α1A, α1B, α1D, α2A,