2-Adrenoceptors in the Eye

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, α2B, α2C,) and species orthologues [13, 14] have been identified (human α2A and rat α2D ) .

G-protein-coupled receptors G-protein-coupled receptors (GPRCs) are the largest family of cell-surface receptors. They mediate the cellular responses to an enormous diversity of signalling molecules, including hormones, neurotransmitters, and local mediators such as proteins, as well as small peptides, light, and odorants. (Fig.1). Other names for GPRCs are heptahelix or seven-transmembrane (7TM) receptors, referring to the conserved structure of the proteins. GPRCs consist of a single polypeptide chain that weaves back and forth seven times across the lipid bilayer of the cell membrane. The amino terminus is located on the outside of the cell and the carboxy terminus on the inside. The ligand-binding domain is buried within the membrane and the ligands that activate the GPRC family are very diverse in size and character ranging from a single photon for the opsins, to large glycoproteins with several hundred amino acids [15]. Membrane-bound receptors transduce a signal from outside the cell, through the plasma membrane, into the cytoplasm of the cell. The cell can respond in several different ways, the responses being mediated by changes in cytoplasmatic signal systems. The GPRC receptors activate guanine- nucleotide-binding proteins (G-proteins) which act on target enzymes such as adenylate cyclase, phospholipase C and phospholipase A2. The second messengers (cAMP, IP3, cGMP, DAG or arachidonic acid) act on protein kinases (PKG, PKA, PKC), or directly on ion channels. The protein kinases act on enzymes, contractile proteins and ion channels in the cell.

10 Light Ca++ Odorants Amino- Proteins acids

NH2 out

Effector Receptor enzymes channels

in COOH G γ D α Intracellular P β messengers

G protein

Figure 1. Schematic model of a G-protein coupled receptor and its signal transmission. A variety of signalling molecules can activate the receptor, which is located in the cell membrane. The GPRC family controls the activity of enzymes and ion channels by catalysing the GDP-GTP exchange on the heterotrimeric G proteins (Gα-βγ)

The GPRCs can be divided into several classes and the adrenergic receptors belong to the rhodopsin-like receptors. This class contains GPRCs with about 300 known genes in different species. Other GPRC classes are the secretin-like family, the metabotropic glutamate-like and pheromone receptors [16].

Adrenoceptors are are subdivided into three families (α1 , α2 and β) according to their pharmacological properties, structure and signalling mechanisms (Fig. 2). Each family contains three subtypes all of which are members of the [13, 17] GPRC superfamily [α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, β3, . The

11 adrenoceptors are proteins of between 402 and 560 amino acids and their endogenous ligands are the cathecholamines adrenaline and noradrenaline [18].

adrenoceptors

α α 1-adrenoceptors 2-adrenoceptors β-adrenoceptors

α α α α α α β β β 1A 1B 1D 2A 2B 2C 1 2 3

Figure 2. Current classification of adrenoceptor subtypes

Classification of α1-adrenoceptors

To date, three distinct members of the α1-adrenoceptor subfamily (α1a, α1b and [19, 20, 21, 22] α1d) have been cloned from different mammalian species .

Unfortunately neither the relationship between the native and recombinant α1- adrenoceptor subtypes, nor the nomenclature used to identify them, has been consistent. In accordance with the IUHPAR [14], there are three subtypes encoded by three genes (α1A / α1a, α1B / α1b and α1D / α1d), where the upper and lower case letters indicate pharmacologically defined and cloned subtypes, respectively. The ’α1c’-adrenoceptor has, after revision of the nomenclature [13, 14, 23] been designated the α1a-adrenoceptor . Abundant expression of all three subtypes is found in the brain of both rat and human, although there is considerable variation in subtype expression between brain regions. All three subtypes also occur in the heart, though α1A is the predominant cardiac subtype in the human. Vas deferens and other smooth

12 muscles appear to express predominantly α1A- and α1D-adrenoceptors. Liver contains predominantly α1B in rat, but α1A in humans. The α1A-adrenoceptor is predominant in the human prostate [24]. Most other tissues produce various combinations of the three subtypes [14, 15, 25]. However, due to a limited number of selective α1D-adrenoceptor substances the α1D-subtype has been difficult to detect by radioligand binding in any native tissue. It seems also that the α1D- adrenoceptor protein is not readily detectable in tissues in which α1D- adrenoceptor mRNA has been described [26]. There is functional evidence of further heterogeneity, particularly for a subtype with low affinity for prazosin [27, 28] . This subtype (α1L) has been suggested to exist in vascular tissues and functionally in the human prostate smooth muscle. However, no gene corresponding to the α1L-adrenoceptor has yet been cloned.

The affinities and selectivities of drugs for α1-adrenoceptor subtypes have been determined in ligand-binding studies. α1A-Adrenoceptor-selective substanses are: WB4101, 5-methylurapidil, niguldipine and oxymetazoline.

Finding selective antagonists for α1B- and α1D-adrenoceptors has been more difficult. Chloroethylclonidine was originally identified as an α1B-adrenoceptor selective substance. However, this drug is less suitable because it inactivates the receptor irreversibly by site-derected alkylation. There has also been controversy over its selectivity in inactivating recombinant α1-adrenoceptor [29, 30] subtypes . Spiperone and risperidone have been shown to be α1B- [31, 32] adrenoceptors selective in the rat . BMY7378 has been shown to be α1D- adrenoceptor selective [33] and this drug has proved useful for clarifying the [34] functional role of the α1D-subtype . The α1a- and α1d-subtypes have been [35] cloned in the pig . The recombinant expression of the α1a-adrenoceptor is distinct, while the α1d-adrenoceptor is evidently weakly expressed (Dr S. Uhlén, personal communication).

13 Signalling mechanisms of the α1-adrenoceptors

The traditional signalling route activated by α1-adrenoceptor subtypes is on the

G protein Gq/11 which activates phospholipase C, and thereby hydrolysis of phosphatidyl inositol 4,5-biphosphate (PIP2). This generates the secondary 2+ messengers inositol 1,4,5-triphosphate (IP3) which releases Ca from intracellular stores, and diacylglycerol (DAG) which synergizes with Ca2+ to [17, 36] activate protein kinase C . All three α1-adrenoceptor subtypes couple to 2+ phospholipase C through the Gq/11 family to increase intracellular Ca (Table 1.1). However, the subtypes have been found to differ in efficiency in activating this pathway [37]. Various other signalling pathways has also been 2+ shown to be activated by α1-adrenoceptors. These include Ca influx (from the extracellular Ca2+), arachidonic acid release, and phospholipase D activation [17, 36] . The predominant ocular sites where α1-adrenoceptors are of relevance are in the radial muscle of the iris, the lacrimal glands and uveal blood vessels.

Physiological responses mediated by α1-adrenoceptors

α1-adrenoceptors mediate some of the main actions of adrenaline and noradrenaline. They regulate numerous physiological processes, such as sympathetic neurotransmission, modulation of hepatic metabolism, control of vascular tone, cardiac contraction, and regulation of smooth muscle activity in the genito-urinary system. It is also clear that α1-adrenoceptors mediate longer term actions of catecholamines, such as cell growth and proliferation [17, 38, 39].

From a clinical point of view, interest in α1-adrenoceptors has focused on cardiac hypertrophy, hypertension, and benign prostatic hyperplasia. Activation [40] of α1-adrenoceptors stimulates a hypertrophic response in cardiac myocytes , and it has been observed that in stress and hypertension, which are serious risk factors for the development of atherosclerosis, catecholamine concentrations are increased.

14 [13, 17, 53, 142, 144 ] Table 1.1. Summary of α1-adrenoceptor subtype characteristics (modified from references and from paper IV).

α1A α1B α1D

Receptor distribution brain, prostate, vas deferens, spleen, kidney, brain, heart, brain, aorta (rat), blood heart, blood vessels blood vessels vessels

Receptor distribution in the iris, ciliary body, retina, iris, ciliary body, retina ? eye choroid (rabbit) (rabbit)

Agonists phenylephrine, phenylephrine phenylephrine 15 oxymetazoline

Antagonists prazosin, 5-MU, WB4101 prazosin, risperidone prazosin, BMY7378

Second messenger system Gq/11 , activation of PLC, elevation of [Ca2+]i, activation of Ca2+ channels

Sensitivity to CEC +/- +++ ++

Abbreviations: CEC, chloroethylclonidine, PLC phospholipase C In smooth muscle and in the heart of both rat and human, several of the α1- adrenoceptor subtypes are coexpressed and it is therefore difficult to distinguish their different functions. According to funtional results, it seems that in the rat the α1A- and α1D-adrenoceptors regulate the larger vessels, [41] whereas the α1B-adrenoceptors control the smaller resistance vessels . The prostate gland in the human expresses α1-adrenoceptors and α1-antagonists have been shown to be effective in relieving urethral obstruction. The [39] predominant adrenoceptor in the human prostate is the α1A-subtype .

Classification of α2-adrenoceptors

Almost fifteen years have elapsed since the α2-adrenoceptor was subdivided into three subtypes: the α2A- α2B and α2C-adrenoceptors. These three receptors are encoded by distinct genes, localized to different chromosomes; in humans [42] [43] the α2A is found on chromosome 10 the α2B gene on chromosome 2 and [44] the α2C gene on chromosome 4 . The subtypes are well conserved in most mammalian species. In rat and mice, however there is a single amino acid

(Cys201 to Ser201) substitution which weakens the affinity of the rodent α2A- adrenoceptor for the classical α2-adrenoceptor antagonists yohimbine and [45] rauwolscine . This receptor has sometimes been defined as the α2D- adrenoceptor but the general consensus is that it is the rodent homologue of the human α2A subtype. α2A- and α2D-Adrenoceptors have never been shown to coexist within one single species. The pharmacological subclassification of α2- adrenoceptors was initially based on the ability of prazosin to inhibit the binding of [3H]yohimbine or [3H]rauwolscine in human platelet and rat neonatal lung. The human platelet receptor was designated α2A (low prazosin affinity) and the rat neonatal lung as α2B (high prazosin affinity). The α2C- adrenoceptor was first found in the opossum kidney-derived cell line (OK- [46] [47] cells) and later in the opossum kidney . The α2C-adrenoceptor is similar to the α2B-adrenoceptor as regards its relatively high affinity for prazosin, ARC239, and spiroxatrine but it has higher affinity for rauwolscine. Studies

16 3 with the highly α2C-selective radioligand [ H]MK912 by Uhlén and Wikberg made it possible to detect even smaller populations of α2C-adrenoceptors in the central nervous system of the rat [48].

Signalling mechanisms of the α2-adrenoceptors

The best-known signalling mechanism of α2-adrenoceptors is the negative [49] coupling to adenylate cyclase via Gi protein . All α2-adrenoceptor subtypes are negatively coupled to adenylate cyclase via Gi (Table 1.2). When Gi is activated by an α2-adrenoceptor, the level of cAMP decreases and protein kinase A is deactivated. In the ciliary process of the eye, stimulation of α2- adrenoceptors conceivably reduces the concentration of cAMP [50, 51]. However, many of the physiological effects of α2-adrenoceptors in tissues cannot be attributed to a decrease in cAMP. Other known signalling mechanisms of α2- adrenoceptors are linked to the following activation: opening of K+ channels leading to hyperpolarization of cells; closing and opening of Ca2+ channels, the latter leading to vasoconstriction. acceleration of Na+/H+ exchange. Stimulation [13, 52, 53] of phospholipase A2 activity leads to arachidonic acid mobilization .

Physiological responses mediated by α2-adrenoceptors

The α2-adrenoceptors are present in virtually every internal organ of the mammalian body, and mediate a wide variety of physiological effects. In the nervous system they act through presynaptic localization and inhibit the release of noradrenaline, acetylcholine, serotonin, dopamine and substance P. α2- Adrenoceptors assist in the modulating sympathetic transmission both in the brain and in the spinal cord, with effects on hypotension, bradycardia, sedation, [52] sleep, analgesia . Peripheral tissues contain presynaptic α2-adrenoceptors on sympathetic nerve endings and postsynaptic and extrasynaptic α2- [52, 53] adrenoceptors on target cells . The α2-adrenoceptors also mediate both contraction and relaxation of vascular smooth muscle. In blood platelets of humans and various other species α2-adrenoceptor activation promotes

17 aggregation. α2-Adrenoceptors are involved in: reduced salivation, and in [54] reduced intestinal secretion and bowel motility . Other known effects of α2- adrenoceptors are reduced insulin release, increased growth hormone release from the anterior pituitary inhibition of lipolysis, inhibition of renin release, increased glomerular filtration [52].

The ability to genetically manipulate α2-adrenoceptor subtypes offers an alternative way to elucidate subtype-specific functions. This has been demonstrated by using either transgenic or knock-out mice. In these studies it seemed that the α2A-adrenoceptors are involved in most, of the classical effects ascribed to α2-agonists: central hypotension, bradycardia, sedation, [55, 56] hypothermia and antiepileptogenic effect . The α2A-adrenoceptors play a major role in regulating basal sympathetic tone. The α2C-adrenoceptors are also involved in modulating central adrenergic and peripheral sympathetic neurotransmission, but the modulation is performed at a lower level of nerve [55] activity . The α2C-adrenoceptor subtype did not play a major role in cardiovascular regulation in the knock-out mouse studies. They might however play a role in regulating of dopamine systems in the brain. They also seem to [55-57] be involved in mediating aggressive behaviour . The α2B-adrenoceptor appears to play a major role in eliciting the vasoconstrictor response to α2- agonists. It is known to be coupled to peripheral pressor responses with hypertension [56].

18 [13, 17, 53, 96,146-148 ] Table 1.2. Summary of α2-adrenoceptor subtype characteristics (modified from references and from paper II ).

α2A α2B α2C

Receptor distribution platelet,brain, spinal cord liver, spleen, kidney, brain, brain, spinal cord aorta, kidney, spleen heart, neonatal lung (rat)

Receptor distribution in the eye iris, ciliary body, retina, choroid iris, ciliary body (human, rabbit) iris, ciliary body (human, (human, cow, pig, rabbit*) rabbit), retina (pig)

Agonists brimonidine, apraclonidine brimonidine, apraclonidine brimonidine, apraclonidine oxymetazoline

19 Antagonists BRL44408 BRL41992 MK912

Second messenger system activation of Gi/0, cAMP ↓ activation of Gi/0, cAMP ↓ activation of Gi/0, cAMP ↓

*α2-adrenoceptor subtypes in rabbit retina and choroid not characterized Vascular α2-adrenoceptors In most mammalian species, contraction of vascular smooth muscle is predominantly mediated via α1-adrenoceptors. At a prejunctional level, α2- adrenoceptors have been found in arteries and veins where they inhibit the release of noradrenaline. However, at a postjunctional level both α1-and α2- adrenoceptors contribute to vasoconstriction in some vascular beds [41, 58-61]. [62, Vasoconstrictor α2-adrenoceptors appear to exist on arteriolar blood vessels 63] [64] and on cutaneous arteries . There are also reports of α2-adrenoceptors on veins in rabbit [65] dog [66] , man [67, 68] and pig [69]. Moreover, bloodvessels have varying sensitivity to sympathomimetic amines, such that some of them may be under the control of circulating catecholamines, whereas others are not. In some vascular beds, the pressor response to sympathetic nerve stimulation is the result of activation of α1-adrenoceptors, whereas the pressor effects of [41] adrenaline are mediated by α2-adrenoceptors . These results were also [70, 71] confirmed in the anterior choroid of the cat . α2-Adrenoceptors have also been located in endothelial cells and cathecholamines are known to cause endothelium-dependent relaxation [72, 73]. It is suggested that one of the relaxant mechanisms depends on nitric oxide release, mediated by the α2-adrenoceptors [74].

The cellular mechanisms underlying the contractile action of α2- adrenoceptor activation are not completely understood. α2-Adrenoceptors are coupled to Gi. However, as there is little evidence to imply that inhibition of the generation of cAMP causes an increase in muscle contracton [52, 75], other mechanisms are probably involved in α2-adrenoceptor-induced contractions. The bulk of the evidence suggests that intracellular Ca2+ is released upon 2+ activation of vascular α2-adrenoceptors, and that intracellular Ca is also dependent on the influx of extracellular Ca2+ [64, 76-78].

However, other pathways have also been suggested, such as α2- [79] adrenoceptor linking to phospholipase A2 (PLA2) in some cell types (Fig 3).

20 When PLA2 acts on phospholipids, they release arachidonic acid, a substrate for eiocosanoid synthesis: this leads to production of a variety of lipid mediators such as prostaglandins, leukotrienes, lipoxins and platelet-activating factor [80]. Arachidonic acid has been suggested to induce contraction in vascular smooth muscle [81, 82] and is also a suggested modulator of K+ channels [83], and Ca2+-sensitivity in vascular smooth muscle.

Phospho α lipase A2 2AR

Gi AA ?

2+ Eicosanoids Ca

Ca2+

Vascular smooth muscle cell

Figure 3. Suggested intracellular mechanisms for α2-adrenoceptors in smooth muscle contraction. α2 AR = α2-adrenoceptor; AA = arachidonic acid; VDCC = voltage dependent calcium channels

21 Ocular α-adrenoceptors Adrenergic regulation of ocular function has been studied intensively because of the ability of the sympathetic nervous system to influence ocular blood flow, pupil diameter, and aqueous humor dynamics. Ocular blood flow regulation Sympathetic nerves deriving from the superior cervical sympathetic ganglion innervate all uveal vascular beds. Two vascular systems supply the ocular tissues: uveal and retinal [84]. The former is densely innervated, while the latter lacks vasoactive nerves. Sympathetic innervation reduces blood flow through all parts of the uvea but has little effect on the retinal blood vessels [85]. It has been suggested that the vasoconstriction induced by the sympathetic nerves in [70, 86] the anterior choroid is mediated by activation of α1 in cat and rat . Adrenaline, however, had a vasoconstrictive effect in the anterior choroid of the cat mediated by both postsynaptic α1- and α2-adrenoceptors, although the predominating effect was caused by the latter [71]. The iris The iris, which controls the size of the pupil and thus regulates the amount of light admitted, is innervated by both sympathetic and parasympathetic nerves. There is a rich adrenergic innervation on the anterior surface of the iris dilator muscle which regulates the contraction of the dilator muscle via α1- adrenoceptors [87-90]. The parasympathetic nerves control the sphincter muscle via muscarinic receptors in various mammalian species [91]. Ciliary body and aqueous humor The ciliary body consists of a central core of muscle fibres and a surface of ciliary processes from which the aqueous humor is produced. The ciliary body is richly, vascularized being supplied by the anterior and long posterior ciliary arteries. Both parasympathetic and sympathetic nerve fibres innervate the ciliary body. The ciliary processes consist of a double layer of epithelium; the outer layer adjacent to the stroma is formed of pigmented cells and the inner layer facing the posterior chamber consists of non-pigmented cells of which the

22 latter are responsible for the aqueous humor formation. Sympathetic innervation is not considered essential for the formation of aqueous humor, but adrenergic agonists can influence on the formation rate [92]. The aqueous humor flows from the posterior chamber through the pupil into the anterior chamber and then drains out of the eye via two pathways. The conventional outflow route which is the principal one, is through the trabecular meshwork, Schlemm´s canal, aqueous veins and the episcleral venous plexus. The other, non-conventional or uveoscleral outflow way is from the chamber angle through the interstitial spaces of the ciliary muscle into the supraciliary and suprachoroidal spaces and out through the sclera [93]. The intraocular pressure (IOP) is dependent on the aqueous humor inflow, outflow, and episcleral venous pressure. Adrenergic drugs can reduce the IOP by influencing in the ciliary epithelium or by regulating the aqueous humor outflow. Both α- and β- adrenoceptors appear to be involved in both these mechanisms. Mixed populations of α1-, α2-, β1- and β2-adrenoceptors have been located in the [94-96] ciliary body . α2- and β2-Adrenoceptors have also been located in the trabecular meshwork [97, 98], in mammalian species. The different adrenoceptor subtypes effect different functions in aqueous humor dynamics. By stimulating

α2-adrenoceptors in the ciliary body, the aqueous flow can be reduced, while the agonist effect on the β2-adrenoceptors is an increase in flow. Consequentely by adding β-blockers, the aqueous humor production can be reduced.

Glaucoma Glaucoma is a common eye disease, that causes optic nerve damage and visual field loss. If allowed to go untreated it can lead to serious visual impairment and even blindness. IOP is usually increased, constituting the major risk factor in developing the disease. In the two main forms of glaucoma - angle closure glaucoma and primary open angle glaucoma - the IOP is increased and is

23 regarded as to be the chief cause of optic disc damage. However, in low-tension glaucoma (where the IOP is normal), the optic disc still suffers damage. Other factors are probably involved in damaging of the nerve. One current theory is that a concomitant vascular disease will increase the susceptibility to nerve damage. In the human eye, the IOP is regulated by a dynamic balance between secretion and outflow of aqueous humor. As high IOP can lead to damage of the optic nerve, with visual field defects as a result and thereby impaired vision, treatment of glaucoma is concentrated on the dynamics of the aqueous humor either by reducing its production or by increasing its effluence. This is achieved by long-term medication, administered topically on the eye. Drugs that have been used for the topical treatment of glaucoma either reduce aqueous humor production, e.g. β-adrenergic antagonists (timolol), α2-adrenoceptor agonists (apraclonidine and brimonidine), carbonic anhydrase inhibitors (acetazolamide and dorzolamide), or enhance the outflow, e.g. analogs of prostaglandin F2α and muscarinic agonists such as pilocarpine.

Adrenergics in glaucoma treatment The use of adrenergic agonists to reduce IOP dates at least far back as 1900, when Darier tried subconjunctival injections of adrenaline [99]. Later (1923) adrenaline was applied topically in glaucoma cases [100]. Following the adrenergic receptor subtype classification and increased knowledge of receptor localization, other adrenergics have been introduced for glaucoma management. Three main groups of adrenergic agents are used for the treatment of glaucoma: adrenaline which is a nonselective agonist for α- and β- adrenoceptors; α2-agonists, and the β-blockers with selective β1-blockers. α1- Antagonists have been undergoing investigation but are not yet used in the glaucoma therapy. Adrenaline (epinephrine) and dipivefrin (a prodrug to adrenaline) are still available for glaucoma treatment. Since adrenaline acts on both aqueous

24 humor inflow and outflow and has affinity for both α- and β-adrenoceptors, the effects on IOP are rather complex. The main effect of adrenaline is considered to be on the aqueous outflow, both the conventional and the uveoscleral routes [101-107]. It is difficult to ascertain which of the adrenergic receptor subtypes is chiefly responsible for this mechanism. Several studies support the involvement of β-adrenoceptors in the effect of adrenaline on outflow facility [108, 109] and the role of cAMP in this mechanism [110, 111]. The β-adrenoceptor [112, 113] was later classified as a β2-adrenoceptor in humans . The effect of adrenaline on the uveoscleral outflow has been suggested to be a secondary effect of the release of prostaglandins, as the IOP reduction induced by adrenaline could be inhibited by oral indomethacin (given in humans) [114]. It [115, 116] [117] has also been suggested that α1-adrenoceptors and α2-adrenoceptors may both be involved in this mechanism. Two hypothetical mechanisms concerning the stimulation of PG synthesis are ( i ) enhanced release of arachidonic acid from the membrane phospholipids by calcium activation of phospholipase A2, or ( ii ) acting as a co-factor for cyclo-oxygenase. However

α1-adrenoceptor antagonists have also been shown to reduce the IOP in experimental animals and humans, at least for a short period of time [118-122]. mainly by increasing uveoscleral outflow. There might also be less specific ways unrelated to the α1-adrenoceptor blocking effect in the stimulation of prostaglandin synthesis, which have not yet been explained [119].

α2-adrenoceptor agonists

Clonidine was the first relatively selective α2-adrenoceptor agonist to find clinical application. However, due to the systemic side effects of clonidine, apraclonidine was developed; this agent is less readily transported across the [123, 124] blood – brain barrier . A newer, more selective α2-adrenoceptor agonist, brimonidine, is now established for the treatment of glaucoma [125]. The main mechanism of action of selective α2-adrenoceptor agonists is believed to be by activating the α2-adrenoceptors on the ciliary epithelium, which reduces cyclic

25 adenosine monophosphate (cAMP) concentrations, causing a decrease in the aqueous humor production [126]. Although the main mechanism underlying the IOP reduction of both apraclonidine and brimonidine is a reduction of secretion, it has been suggested that they may also have some effect on uveoscleral outflow, but the detailed mechanisms are not yet completely clarified [127, 128]. Brimonidine has furthermore been shown in animal models to reduce secondary degeneration of the optic nerve. The exact mechanisms of the neuroprophylactive effects of α2-adrenoceptor agonists are still hypothetical, but activation of the α2-adrenoceptor by brimonidine has been shown to up- regulate anti – apoptotic genes [129] and clonidine increased the expression of bFGF mRNA- in rat photoreceptors [120].

26 AIMS OF THE PRESENT STUDY

• to characterize pharmacologically α1-adrenoreceptor subtypes in porcine and rabbit eyes

• to characterize pharmacologically α2-adrenoreceptor subtypes in the four main porcine eye tissues: iris, ciliary body, retina, and choroid

• to study the effect of α2-adrenoceptor agonists on porcine ciliary artery

• to characterize the α2-adrenoreceptor subtypes mediating vasoconstriction of porcine ciliary artery

27 Figure 4. Schematic picture of eye anatomy.

28 MATERIALS AND METHODS A short presentation of the methods used in this thesis is given below. Complete descriptions are given in the separate papers.

Animals In all five investigations tissues from the pig generally and from the pig eyes were used. The reason for choosing pig eyes was simply because they are quite similar to the human eye. They are of the same size and both human and pig eyes are similarly pigmented. The pig eye is readily obtained from the slaughterhouse. In paper IV, albino rabbit eyes were chosen instead, due to a difficulty in analysing the pigmented parts of the pig eye.

Tissue preparations for binding studies Membrane preparations from pig tissues(I,III) Various pig tissues were obtained from the local slaughterhouse. These were cut into smaller pieces and homegenized immediately or frozen to -80°C within 1h and then stored until homogenization. Tissues were homogenized in ice- cold 50 mM Tris-HCL, 5 mM EDTA, 1 mM PMSF (phenyl methyl sulphonyl fluoride), 10 µg /ml soybean trypsin inhibitor (STI) and 200 µg /ml bacitracin, pH 7.5, using a motor-driven teflon glass homogenizer. The homogenates were spun at 500 x g, and supernatants were collected and spun at 38,000 x g for 30 min. The pellets were then washed once with the same buffer and then with a buffer containing a lower concentration of EDTA (1.5 mM). The final pellets were diluted to about 1:8 times weight. Eye tissue preparations (II, IV) Pig eyes and rabbit eyes were obtained from local slaughterhouses. The eyes were dissected under a dissecting microscope where the iris, ciliary body, choroid and retina were excised. The tissues were then prepared as described for the other tissues, except those from the rabbit eye, where the

29 homogenization was done manually. In the preparations from the pig eyes the pellets contained a sediment of melanin, which was discarded and only the soft top layer of membranes was used.

In vitro studies on ciliary arteries (V) Fresh pig eyes were obtained from the local slaughterhouse. All dissections of the eyes and the mounting of blood vessels in the small vessel myograph were done under the dissecting microscope. Ring segments (∼ 2 mm) were mounted on 40 µm wires in a small vessel myograph for isometric recording (J.P.Trading, Denmark) (Fig. 5).

Figure 5. Photograph of small vessel myograph and schematic drawing of a blood vessel mounted in the bath.

The experiments were performed in physiological salt solution (PSS). The preparations were allowed to equilibrate in oxygenated (95% O2 and 5% CO2 )

30 PSS at 37°C, pH 7.4, for about 30 min. The vessels were stretched by using a force corresponding to 1.5 – 2.5 mN. The vessels were used either immediately or saved until next day (and used within 36 h). They were kept in PSS in a refrigerator until used. After normalization, the contractile ability of the vessels was tested by stimulating the arterial rings with 125 mM K-PSS, which was given until reproducible responses were recorded. After washout, the preparations were exposed to noradrenaline (10-6 M) and allowed to contract for about 5 min. Contractility was also tested with carbamylcholine chloride (10-6 M), and phenylephrine (10-6 M). PSS contained propranolol (10-6 M) to inhibit possible activity from β-adrenergic receptors and indomethacin (2.8 x 10-6 M) to prevent synthesis of endogenous prostaglandins. To obtain the optimal response from a vessel mounted in a small vessel myograph, its internal circumference of the vessel, the wall length, and tension were calculated. The effective pressure, P can be determined by the Laplace relation:

P = wall tension / (internal circumference/(2 x π)).

The internal circumference, usually denoted IC100 (L100) of the vessel, which is the tension the vessel would have if relaxed and subjected to a transmural [131] pressure of 100 mmHg can be determined . However, the factor IC100 varies between the preparations. In the porcine ciliary arteries, the relationship between resting wall tension and the internal circumference was determined, and the internal circumference, IC60 (L60), corresponding to a transmural pressure of 60 mmHg for a relaxed vessel was calculated. The level of optimal passive tension for the ciliary arteries had been determined in preliminary experiments (n=3) and defined as that tension at which the contraction to 125 KCl mM was maximal.

31 Analysis of binding data (I-IV): Some basic formulae have been used in the analysis of the binding experiments. The law of mass action This is the fundamental principle in all interactions between ligand and receptor where it is assumed that each ligand (L) binds to each of the receptors (R) in a reversible manner. Kaff = affinity constant. Kd = dissociation constant.

[LR] L+R LR K = K = 1 aff [L][R] aff Kd Kaff

For computer modelling, the following formula was used:

n Kib Fi Rb Σ m + Ni Fi Bi = b=1 Σ 1 + Kab Fa a=1

This describes the binding of the ligand i (Bi) to all receptors in the assay, in the presence of (any number of) competing ligands (a)

Kib and Kab are the affinity constants for ligands “i” and “a” for receptor “b” respectively. Fi and Fa are the free concentrations of ligands “i” and “a”, respectively. Rb is the concentration of receptor site “b”, and Ni the non- specific binding for ligand “i”. The free concentrations of ligands “i” (Fi) could be estimated from the total concentrations of ligands (Ti) by numerically calculating : f(Fi) =Ti-Bi-Fi = 0 Computer modelling of the data was performed using a radioligand binding analysis package (Wan System, Umeå, Sweden) on a MacIntosh computer. The data were fitted to equations assuming that the ligands bound reversibly to independent sites according to the law of mass action [132, 133], and using non – linear least squares regression [134]. The same set of data were fitted

32 either into a one-site model or to a two-site model, assuming the presence of either one or two independent binding sites. An F-test comparing the sums of squares for the different models was then used to judge which model approximated the data best [132]. The binding constants of competing drugs are given as dissociation constants in papers (I) and (II) as Kd in papers (II) and (IV) as pKi values. (See also Uhlén et al. [135])

Analysis of the dose – response studies (V)

Hill equation The concentration – response curves were fitted to the Hill equation and calculated by non-linear regression using GraphPAD Prism Program (Graph PAD Software, San Diego, CA). Sensitivities to drugs were calculated on the basis of data from individual vessels and are expressed as EC50, i.e. the agonist concentration needed to produce 50% of the maximum response.

Response = E0 + Emax- E0 1+ [A] n

EC50

E0 denotes the minimum response and Emax the maximal response. A is the concentration of agonist. n is the Hill coefficient (slope of the function).

33 Schild plot When antagonists produced parallel displacements of agonist concentration – response curves, Schild analysis was constructed by means of least – squares linear regression of log (CR-1) versus log antagonist concentration, where CR is the concentration ratio of the agonist in the absence and presence of antagonist [136]. log (CR-1) = log ([A]) – log (Kb)

Concentration ratios were calculated at the EC50 level. Provided that the regression of the Schild plot is linear and that the slope does not diverge significantly from unity, the pA2-value, which is the intercept on the abscissa of the Schild plot, is equal to the negative logarithm of the equilibrium constant for the antagonist, then pA2 = – logKB = pKB.

The slope was also constrained to unity and the pKB value was calculated. The solver function of Microsoft Excel was used to fit the model of linear regression.

Drugs and chemicals Some comments are necessary here regarding the drugs used in this thesis: The radioligands used in the binding studies (I and II) were [3H]RX821002 and [3H]MK912. [3H]RX821002 is an imidazoline with high affinity for all three 3 α2-adrenoceptor subtypes. [ H]MK912 is a yohimboid compound with high affinity for the α2C-adrenoceptor. The ranking of affinities was α2C >α2A ≥α2B 3 (I). [ H]prazosin, a quinazoline was used to label α1-adrenoceptors in both pig and albino rabbit tissues (III and IV). [3H]prazosin does not distinguish between the different α1-adrenoceptor subtypes. Chloroethylclonidine, which was used in paper (IV), is an α1-adrenoceptor subtype selective alkylating agent that inactivates irreversibly the α1B-adrenoceptor more efficiently than it [29] inactivates the α1A- or α1D- adrenoceptor subtypes .

34 Brimonidine and apraclonidine are α2-selective agonists which were used in paper (V), where brimonidine has higher affinity for α2-adrenoceptors than apraclonidine [125]. Phenoxybenzamine, used in some experiments in (V), is an

α1- and α2- antagonist that binds irreversibly to both α1- and α2-adrenoceptors [137] but has higher affinity to α1- than to α2-adrenoceptors .

Statistical analysis

EC50-values of the agonists were analysed by the one-way analysis of variance (ANOVA) and by Bonferroni´s multiple comparison test for comparison of groups in (V). P-values less than 0.05 were considered significant.

35 RESULTS

Identification of α2-adrenoceptor selective drugs in pig (I) The intension in paper I was to evaluate the subselectivity of ligands, for the

α2A-, α2B- and α2C-adrenoceptors in pig. Radioligand binding studies on rodents have shown that the α2A- and α2C-adrenoceptors coexist in the central [48, 138] nervous system and α2A- and α2B-adrenoceptors in kidney . In the present study the cerebellum and the kidney cortex proved to be useful tissues for the characterization of α2-adrenoceptor subtypes also in the pig. In pig cerebellum 3 the α2-adrenergic antagonist [ H]MK912 was applied in saturation and competition experiments, whereas [3H]RX821002 was a better choice to label

α2-adrenoceptors in the kidney cortex by virtue of its lower non-specific binding in this tissue. In pig cerebellum, both α2A- and α2C-adrenoceptors were identified (91 % and 9 % respectively) and in kidney α2A- and α2B- adrenoceptors were localized (48 % and 52 % respectively). The Kd values for

α2A- α2B- and α2C-adrenoceptors of 9 drugs were evaluated (Table 2 paper 1).

For example BRL44408, was found to be α2A-selective; BRL41992 was found to be α2B-selective; and MK912 was α2C-selective. These data suggest that a minimal set of these drugs would suffice to distinguish between the different

α2-adrenoceptor subtypes.

α2-adrenoceptors in pig eye (II)

In paper II the α2-adrenoceptor subtypes in different parts of the pig eye were investigated: in the iris, ciliary body, retina and choroid. In paper I the affinities of the radioligands and competitors suitable for distinguishing between the porcine α2-adrenoceptor subtypes had been determined, and most of these drugs were also used in eye tissues. In the choroid, the ciliary body and the retina, [3H]MK912 was used as labelled ligand; while in the iris [3H]RX821002 was used because of its lower non-specific binding. All the competition curves

36 constructed for iris, ciliary body and choroid were monophasic, indicating the presence of only one receptor population. The Kd-values of the competing drugs corresponded best for an α2A-adrenoceptor (Table III paper II). The choroid contained high densities of α2A-adrenoceptors (900 fmol/mg) whereas in the ciliary body they were lower (230 fmol/mg) and in the iris, they were estimated to 87 fmol/mg protein. 3 In pig retina the α2C-selective radioligand [ H]MK912 labelled two receptor populations, conceivably α2A- and α2C-adrenoceptors. This conclusion is based on the following facts. First, the competition curve of the α2A-selective competitor BRL44408 was biphasic. Second, when adding a fixed, predominantly α2A-blocking concentration of BRL44408 (100 – 167 nM) to the competition experiments, the affinities of drugs selective for α2B- and α2C- adrenoceptors in the masked competition curves correlated best to the α2C- adrenoceptor. Finally when correlating the affinities from retina with those in pig cerebellum and kidney cortex this suggestion was further confirmed (Figs

4 and 5, Table III, Paper II). The receptor densities of α2A- and α2C- adrenoceptors were 20 and 3.6 fmol/mg protein respectively.

α1-adrenoceptor subtypes in the pig and rabbit eyes (III, IV)

In paper (III) the identities of the α1-adrenoceptor subtypes were studied in various pig tissues, the intention being to identify drugs subselective for the α1- adrenoceptor subtypes. The experiments were performed by using [3H]prazosin as labelled ligand. From cerebral cortex, heart and aorta, two α1-adrenoceptor populations were classified: α1A and α1B. In liver only the α1A-adrenoceptor was identified. In the adrenal gland the same drugs were tested as in the cerebral cortex and liver. All the competitors showed fairly low affinities in the adrenal gland, except for prazosin (Table 1, Paper III). 3 Having the information on the pKd values of [ H]prazosin obtained from saturation experiments and the affinities of the drugs in porcine tissues (III) it

37 was possible to initiate the investigations into the α1-adrenoceptor subtypes in the pig eye (IV). At an early stage the experiments with [3H]prazosin in the pigmented tissues of the pig eye showed extremely high non-specific binding (60-90 %). Even in the retina the small amounts of retinal pigment disturbed the analysis, the cause presumably being the properties of melanin in the pigmented tissues. The capacity of melanin to bind and accumulate substances is well known [139]. Chloroquinephosphate was therefore tested in the present experiments in an attempt to diminish the unspecific binding. In order to ascertain the appropriate dose to block melanin (but not the α1-adrenoceptors), competition experiments with [3H]prazosin and chloroquinephosphate were performed in pig choroid (high melanin content) and cerebral cortex (low melanin content) From the competition curves obtained from the choroid membranes it was evident that 3 µM chloroquinephosphate did prevent most of the non-specific binding of [3H]prazosin (Fig. 6).

100

50 H]-prazosin bound % 3 [

0 -8 -7 -6 -5 -4 -3 -2 Chloroquinephosphate log (M)

Figure 6. Competition curves of chloroquine phosphate in various concentrations in pig cerebral ( ) and pig choroid (■) membranes, using [3H]prazosin as labelled ligand.

38 Chloroquinephosphate was then accordingly added to the membranes before the ligand and competitor experiments were performed. This experimental design succeeded best in the retina, where the non-specific binding was halved (from 60% to about 30%). The non-specific binding in iris, ciliary body and choroid was however still too great to allow detection of any specific α1-adrenoceptor binding. To avoid this problem caused by the melanin, the investigations were extended and experiments on the albino rabbit eye were included. [3H]prazosin was used as labelled ligand in all competition experiments, in both rabbit eye membranes and pig retina membranes. In the experiments on pig retina, the following competing drugs were used: WB4101, with high affinity for α1A- and

α1D-adrenoceptors; 5-methylurapidil, with high affinity for the α1A- and low affinity for both α1B- and α1D-adrenoceptors, BMY7378 and SKF104856, which in other species have been shown to possess high affinity for the α1D- adrenoceptor [25]. The competition curves of 5-methylurapidil and WB4101 were biphasic thus identifying two α1-adrenoceptor-sites in the pig retina. The high-affinity site corresponded best to the α1A-adrenoceptor, while the second, low-affinity site corresponded best to α1B. In iris, ciliary body and retina of the albino rabbit, 5-methylurapidil and

WB4101 showed biphasic competition curves and pKi values of the two receptor sites corresponded closely to α1A- and α1B-adrenoceptors respectively. In the rabbit choroid, 5-methylurapidil and WB4101 showed high affinity in monophasic competition curves and the conclusion was that only the α1A- adrenoceptor subtype was detected here.

To further verify the two sites as being receptors for α1A- and α1B- adrenoceptors, the membranes from iris, ciliary body and retina were preincubated with 10 µM of the irreversible α1B-adrenoceptor blocker chloroethylclonidine (CEC) [29]. After CEC treatment the competition curves of 5-methylurapidil became monophasic and the remaining high affinity sites corresponded closely to the α1A-adrenoceptor (Table IV, paper IV).

39 Effect of α2-adrenoceptor agonists in the ciliary artery (V)

In paper (V) the effect of some selected α2-adrenoceptors agonists on the porcine ciliary artery was investigated. Three α2-adrenoceptors agonists were chosen: apraclonidine, brimonidine and oxymetazoline. All α2-adrenoceptor agonists proved to be potent vasoconstrictors in the pig ciliary artery and the

EC50 values obtained from the concentration – response curves were ranked as follows (expressed in nM): brimonidine 2.1, oxymetazoline 5.3 and apraclonidine 13.0. In order to identify the subtypes of α2-adrenoceptors mediating the vasoconstrictive response of the pig ciliary artery, concentration- response curves for brimonidine were constructed in the presence of the following antagonists: BRL44408 which is selective for the α2A-adrenoceptor,

ARC239 which shows low affinity for the α2A-adrenoceptor and high affinity for the α2B- and α2C-adrenoceptors, and prazosin which shows low affinity for the α2A-adrenoceptor and high affinity for the α2B- and α2C-adrenoceptors. The concentration – response curve for brimonidine was shifted toward the right and parallel for all antagonists, indicating competitive antagonism. Significant parallel displacement occurred at high concentrations of ARC239 and prazosin both of which are antagonists with low affinity for the α2A-adrenoceptor and high affinity for α2B-and α2C-adrenoceptors. Analysis of the data by Schild regression gave pA2 values for BRL44408, 7.85±0.12; ARC239, 5.86±0.12; and for prazosin, 6.02±0.07.

40 DISCUSSION

α1-Adrenoceptors in the eye

Information regarding α1-adrenoceptor subtype distribution in the eye obtained in earlier studies is scattered and incomprehensive and systematic studies to identify α1-adrenoceptor subtypes in the various structures of the eye are lacking. In the iris, however α1-adrenoceptors are well known and also the most frequentely studied part of the eye. As mentioned in the introduction, the iris dilator muscle is innervated by sympathetic nerve fibres effecting the α1- [88-90, 140] adrenoceptors . In the present study (IV) both α1A- and α1B- adrenoceptors were found in the rabbit iris, thus agreeing with studies using PCR techniques in rat iris [141] and with binding studies and mRNA analysis [142] rabbit iris . The α1B-adrenoceptor has also been immunohistochemically localized in the iris dilator muscle, and ciliary processes (epithelial layers) of [143] the rat . On a functional level, it is not entirely clear which of the α1- adrenoceptor subtypes is involved in contracting of the dilator. In the rat iris the [144] α1B-adrenoceptor is believed to be responsible for dilating the iris , whereas it is suggested that in rabbit iris a putative “α1L-adrenoceptor” is responsible for the contraction of the iris dilator [142]. However, it has also been proposed that [23] the α1L-adrenoceptor is a conformational state of the α1A-adrenoceptor and it has been shown that human cloned α1A-adrenoceptor isoforms display the α1L- adrenoceptor pharmacology in functional studies [145]. The information from earlier work on the pharmacological identity and distribution of α1-adrenoceptor subtypes in the ciliary body is less extensive because fewer studies have been performed here. Greatest interest has been concentrated on the effect of α1-adrenoceptor agonists and antagonists on IOP and aqueous humor dynamics. In several studies, α1-adrenoceptor antagonists have been shown to lower the IOP by increasing the uveoscleral outflow in the

41 [118, 119] rabbit . It is not known if this mechanism is coupled to a specific α1- adrenoceptor subtype. Sympathetic vasoconstriction in the anterior choroid of rat and cat is believed [70, 86] to be mediated by α1-adrenoceptors . In the present study it was shown that the rabbbit choroid α1-adrenoceptor subtype is of the α1A-subtype.

In studies (III and IV) no α1D-adrenoceptors were detected in the porcine tissues. In other species the drug BMY7378 has been shown to be α1D- selective. In the rat, rabbit adrenal gland, aorta and vas deferens have shown [25, 41] populations of α1D-adrenoceptors . In the present studies (III and IV), however, BMY7378 showed only low affinity in all tissues where α1D- adrenoceptors would be expected. At the time for the present work the only

α1D-adrenoceptor selective substance was BMY7378, and this might not be selective in pig tissues. Another more plausible explanation is that the α1D- adrenoceptor is not widely expressed in pig tissues.

α2-Adrenoceptors in the eye

In earlier studies using radioligand binding techniques α1- and α2-adrenoceptor subtypes were localized in the iris /ciliary body of the albino rabbit eye [94].

Later α2A-adrenoceptors were identified as an isolated population in the albino rabbit ciliary body [96]. In the present study where pig eyes were used we wanted to extend the investigations into α2-adrenoceptor subtypes to the choroid and retina as well as the ciliary body and the iris. One remarkable finding was the high density of α2A-adrenoceptors in the choroid. The importance of this is not fully understood, but they might play an important physiological role in the choroid. In all uveal tissues, i.e. iris, ciliary body and choroid, only α2A-adrenoceptors were found. Later similar radioligand-binding studies in cow and human, performed by other investigators, only α2A- adrenoceptors were localized in the iris, ciliary body choroid and retina [146-148]. These findings are in agreement with study (II) except in the pig retina where

42 two populations of α2-adrenoceptors were characterized (85% α2A- and 15%

α2C-adrenoceptors) in our study. Various reasons for this could be discussed. An important difference between study (II) and other studies [96, 146-148] is the 3 advantages of the α2C-selective radioligand [ H]MK912 used in the pig retina over radioligand [3H]RX821002 which was used in the bovine and human 3 retinal membranes. [ H]RX821002 is slightly α2A-selective and therefore an inferior choice to label smaller populations of α2B- or α2C- subtypes. The 3 excellent properties of [ H]MK912 to label smaller populations of α2C- adrenoceptors is known from earlier studies by Uhlén et al. [48] and from our study (I). A further advantage of the experimental design, where we used a small amount of an α2A-selective blocking drug in some of the competition curves, helps to distinguish the binding affinity of the tested drugs for the labelled receptors. This experimental design also renders the non-α2A-subtypes more visible, as they become the majority of the labelled sites.

To date, the α2-adrenoceptors in the eye have been investigated by several techniques. In autoradiographic studies, high concentrations of α2- adrenoceptors were found in the epithelium of both iris and ciliary body, as well as in the ciliary muscle, retina and RPE of the human eye [149]. In these studies, the total pool of α2-adrenoceptors was visualized, but the individual receptor subtypes were disregarded. Immunofluorescence labelling of the human ciliary body indicates the presence of α2B- and α2C-adrenoceptor subtypes, but not the α2A-subtype, while in the rabbit all three subtypes were localized [153]. In cultured human trabecular meshwork cells, however, only [151] α2A-adrenoceptors were localized by immunocytochemistry . It is of interest that the immunofluorescens technique identifies all three subtypes in the rabbit ciliary body [150], whereas the radioligand binding detects [96] only α2A-subtype . Because the immunofluorescence technique is not quantitative, there might be very low concentrations of α2-adrenoceptor subtypes in the ciliary body of the rabbit that are not detectable with

43 radioligand binding. It is surprising that no α2A-adrenoceptors were detected in the human ciliary body by immunohistochemistry, while the only subtype detected by radioligand binding was the α2A-subtype. One reason for this could be that while the radioligand - binding technique detects binding in the tissue as a whole, only small areas of the ciliary body are studied by immunohistochemistry. In the present study (II), however, only α2A- adrenoceptors were detected in the iris, ciliary body and choroid. This indicates that the predominant population is the α2A-subtype, as smaller populations down to ~ 10 % would have been detected by [3H]MK912, at least in the choroid and the ciliary body. The concusion is though, that despite some discrepancies in different techniques between the investigations by other authors and our study (II), it is highly likely that the quantitatively predominating subtype in the eye from several mammalian species (bovine, porcine and human) is the α2A- adrenoceptor.

The results of paper (II) indicated large amounts of α2A-adrenoceptors in the vascular parts of the eye, mainly in the ciliary body and choroid. The first objective of study (V) was therefore to investigate the effect of α2-adrenoceptor agonists on the porcine ciliary artery which nourishes the uvea and to characterize the functional α2-adrenoceptor subtypes. All the proved α2- adrenoceptor agonists showed to be potent vasoconstrictors, indicating the presence of postjunctional α2-adrenoceptors in the porcine ciliary artery.

The presence of α2-adrenoceptors in the ocular vessels of mammalians is not a clear-cut fact. There are several aspects to take into consideration when evaluating the results of other studies concerning the presence of α2- adrenoceptors in the ocular vessels: 1) type of vessel studied. 2) techniques used to study the effect of drugs on ocular vessels. 3) the myogenic tone of the vessel when adding the α2- adrenoceptor agonists. 4) the species variation and consequently the presumed

44 variation in distribution of α2-adrenoceptors in ocular tissues between different species.

The regional differences in α2-adrenoceptor distribution between the different parts of the vascular bed are well known [58, 61]. Several studies on resistance arteries such as smaller precapillary arteriolae have shown [63, 152] pronounced contractions in response to α2-adrenoceptor stimulation . It is suggested that there is a correlation between the diameter of the vessel and the response by α2-adrenoceptor agonists in human where best responses of α2- adrenoceptor agonists were induced in vessels with a diameters in the range 100-400 µm [62]. Vessel diameters in the present study on pig ciliary arteries was measured to be 247 ± 27 µm. Moreover in vitro studies with the myograph [153] technique verified α2-adrenoceptors also in the the bovine ciliary artery .

The existence of α2-adrenoceptors in the retinal vessels is an important question. As α2-adrenoceptor agonists are used in glaucoma therapy, vasoconstriction would be an unwanted side effect, especially in the retinal circulation. In bovine retinal vessels α2-adrenoceptors have been shown by radioligand binding [154, 155]. However preliminary studies in our laboratory have failed to show functional α2-adrenoceptors in the bovine retinal vessels (Fig. 7).

45 Figure 7. Original trace recordings of brimonidine at increasing concentrations in the bovine retinal artery. The experiments were performed in the presence of 10-6 M propranolol and 2.8 x 10-6 M indomethacin. Na=noradrenaline; w=wash. Brimonidine induced vasoconstriction -7 at a high concentration (10 M) of brimonidin, indicating that only α1-adrenoceptors were activated.

The presence of α2-Adrenoceptors in the human retinal vessels have not unambigously been shown in functional studies or in in vitro studies [156, 157].

Furthermore, no effect on the retinal vessels after topical treatment with α2- adrenoceptor agonists was shown by non-invasive techniques [158, 159]. It is known that the concentration of brimonidine in the vitreous humor of the monkey eye attains 100-170 nM after topical treatment [125]. Since these concentrations are above the level of ∼ 2 nM, any possible retinal α2- adrenoceptors should be activated [160]. However, the influence on retinal circulation has not been studied in aphakic or vitrectomized patients where concentrations of brimonidine were measured to 185 ± 500 nM intravitreally[161].

One possible explanation why α2-adrenoceptor agonists induce a powerful vasoconstriction in isolated ciliary arteries, but not in the retinal

46 vessels, could accordingly be that the ciliary arteries contain larger amounts of

α2-adrenoceptors than other, smaller vessels in the eye. Retinal vessels which have a diameter of 30-35 µm do not strictly belong to the resistance vessels. Variability in myogenic tone of the vessel segments could be another explanation for the variable response elicited by α2-adrenoceptors in eye arteries in vitro. In several studies, α2-adrenoceptors were only activated in the presence of a slight increase in vessel tone [77, 153]. In the present study (V), however, raising basal tone in the pig ciliary arteries by increasing the extracellular K+- concentration did not affect the contractile responses induced by brimonidine, compared with responses obtained in physiological buffer.

The second important finding in Paper V was that the α2A-adrenoceptors mediated the vasoconstriction in the examined intraocular part of the pig ciliary artery. The Kd-values (in this study recalculated to pKi-values) obtained from the ligand binding studies in Paper II gave an accurate guide to the affinities of the antagonists at the respective subtypes of receptor. Comparision of the pKB values of the BRL44408, ARC239 and prazosin obtained from the Schild analysis, versus the affinities obtained in binding experiments implies that the

α2A-adrenoceptor is responsible for the contraction induced by brimonidine (Table 2 Paper V).

47 CONCLUSIONS

• The predominating α2-adrenoceptor subtype in the ocular tissues of the pig

is the α2A-subtype. In the iris, ciliary body and the choroid, only α2A-

adrenoceptors could be identified. In the pig retina, most were α2A- while a

small proportion of α2C-adrenoceptors was detected.

• Mixed populations of α1A- and α1B-adrenoceptors were identified in the pig retina, and also in the ocular tissues of the albino rabbit.

• The α2-adrenoceptor agonists brimonidine and apraclonidine are potent vasoconstrictors in the porcine posterior ciliary artery.

• The α2A-adrenoceptor subtype mediates the vasoconstriction induced by brimonidine in the porcine posterior ciliary artery.

48 IMPLICATIONS FOR FUTURE RESEARCH

The previous classification of adrenoceptors into α1, α2, β1 and β2 has been a prerequisite for developing selective drugs for ocular therapy. These drugs are mainly used to reduce intraocular pressure in glaucoma. The purpose of this thesis is to increase understanding of the distribution of the α1- and α2- adrenoceptor subtypes in the eye and their involvement in physiological processes, to achieve new strategies for drug development. Although the present studies offer a better insight in the characterization and distribution of

α1- and α2-adrenoceptor subtypes, some questions remain unanswered.

Vasoconstriction and pupil dilation are well known α1-adrenoceptor [91, 165, 166] effects in the eye . The α1-adrenoceptor agonists phenylephrine, oxymetazoline and naphazoline are used either for pupil dilation in clinical examinations or for local application in order to induce vasoconstriction in the conjunctiva. These substances sometimes cause unwanted cardiovascular side effects. The information regarding α1-adrenoceptor subtypes mediating vasoconstriction in humans in general, is still scarce and to date, no studies have been performed on ocular vessels. It is also unknown which α1- adrenoceptor subtype that is responsible for the pupil dilation in the human. If different subtypes are involved in pupil dilation and vasoconstriction this could have implications for developing subselective drugs.

α1-Adrenergic agents have no place in glaucoma treatment today. One main reason is adverse systemic effects. As shown in paper IV, α1A- and α1B- adrenoceptors are located in most of the examined parts of the rabbit eye with exception of the choroid, where only α1A-adrenoceptors were detected. However, further studies are needed to locate the subtype distribution on a cellular level. As it is suggested from earlier studies, α1-adrenoceptors are involved in IOP-reduction, partly through endogenous prostaglandin synthesis, it would be interesting to further investigate which α1-adrenoceptor subtypes

49 that are located in the trabecular meshwork, Schlemm´s canal, and the area which is connected to the uveoscleral outflow pathways. Further investigations are also needed to penetrate the detailed mechanisms behind IOP-regulation and to evaluate the possibility of reducing systemic side effects via use of subselective α1-adrenoceptor drugs. Several studies by other authors and ours (Paper II), implicate that the predominating α2-adrenoceptor subtype in the mammalian eye is the α2A- subtype. If different α2-adrenoceptor subtypes had been responsible for the IOP regulation and vasoconstriction this would have stimulated development an α2- adrenoceptor subselective drugs for glaucoma treatment. It seems more probable though, that the same subtype is responsible for all known classical

α2-adrenoceptor mechanisms in the eye. In light of these facts, the use of more subtype-selective α2-agonists is of little interest. On the other hand, it is not inconceivable that small populations of α2B- and α2C-adrenoceptor subtypes are present in ocular tissues, but their specific functions in the eye are unknown.

In retrospect, the development of more selective α2-agonists with higher affinity for α2-adrenoceptors has proved beneficial in general terms. More selective agonists could be preferable in the perspective that receptor-mediated agonists could reduce systemic side effects, which still is a problem with the available α2-agonists. The fact that a major part of a drug adminstered topically is absorbed systemically, while only minor concentrations reaches the eye tissues calls for developing drugs with minimal systemic side effects [167]. An unexpected effect of α2-adrenoceptor agonists has been to reduce retinal cell [132, 168] ganglion death in rat models . It is unknown to which α2-adrenoceptor subtypes these mechanisms are coupled; if such a neuroprotective effect exists in the human retina or if this is of relevance to the human glaucoma eye. These matters remain for future investigations.

50 ACKNOWLEDGEMENTS

This thesis was undertaken at the Department of Pharmaceutical Biosciences, Faculty of Pharmacy, and the Department of Neuroscience, Divisions of Pharmacology and Ophthalmology, Faculty of Medicine, Uppsala University.

I wish to express my sincere gratitude to the following persons:

Professor Albert Alm, Department of Neuroscience, Ophthalmology Uppsala University for unreserved support of this project from inception to its conclusion, and for valuable scientific discussions;

Professor Jarl Wikberg, Department of Pharmaceutical Biosciences, for your professional guidance through pharmacology research, for encouraging independent work, and for the facilities placed to my disposal at your laboratory;

Associate Professor Staffan Uhlén my supervisor and co-author at the Department Pharmaceutical Biosciences, for sharing your comprehensive knowledge of pharmacology and teaching me all those practical and theoretical details about radioligand binding;

Professor Johan Stjernschantz at the Department of Neuroscience, Pharmacology Division, for generously allowing me to use the myograph at your lab and for valuable discussions in experimental eye research. Many thanks for your encouraging attitude and for including me in your research group without reservations, in spite of the fact that my project was ”outside”;

Associate Professor Ulf Simonsen, Department of Pharmacology, University of Aarhus, Denmark, for fruitful collaboration; colleagues at the Department Pharmaceutical Biosciences: Rutha, Maija, Peteris, Jonas, Helgi, Per-Anders, Yun, and all other past and present members in the group, for creating such an inspiring scientific environment; all my friends in the ”Pharmacia group” at the Neuroscience Department, for including me in your group and for making my time at the Department enjoyable and such fun; Eva Kumlin for teaching me how to use the small- vessel myograph and for valuble help and discussions; Bahram Resul for solving (!) chemistry problems, both theoretically and in practice; Henrik Jernstedt for computer help, Irene Aspman, Kerstin Bergh, Kiyo No, Parri Wentzel, Sofia Mikko, Niyun Jin and Therese Lifvendahl for all support and assistance; Maria Astin (Astra-Zeneca) for good advises and for lending pictures to the manuscript;

51 the groups of Lars Oreland and Dan Larhammar in the ”neighbouring corridor”, for providing a nice atmosphere; Earl for lingustic corrections of the manuscript and Robert for computer help.

I would also thank all colleagues at the Department of Ophthalmology and especially: Olav Mäepea for always lending a helping hand with computer problems and for skilful poster work; Birgit Andersson, for professional help and for contributing to the pleasant atmosphere at the Department; Lill-Inger Larsson for cheering me up and for the fun we had at the ARVO meetings.

To all colleagues at the Eye Clinic, heartful thanks for being so patient with my absence from the Cataract Division. In this respect I would especially like to thank Virpi Khalifeh-Barke and Pekka Niemilä for your ability to appreciate my predicament and so generously accept the consequences for the Clinic.

Editor Max Brandt for excellent linguistic revision of the manuscript

Many thanks to my friend Eva Baecklund for sharing my worries and frustrations over these years and for the innumerable conversations about clinical work, research and life in general during our evening walks; my friend Anna-Stina Ponsford for supporting me during this past year. all other friends, not mentioned but not forgotten, for good times, for laughs and jokes and for parties and recreation;

To my dear children Jan and Agnes, thank you for being just what you are and giving me joy and meaning in my life.

Olle my beloved husband, thousand thanks for never-failing support and understanding, for always being interested in and listening to any problem in my research project, and for sharing both joys and sorrows in life. Without you this thesis would never have been completed.

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