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210 British3JournalofOphthalmology, 1992,76,210-213 Localisation ofprostaglandin F2, and E2 binding sites in the human eye Br J Ophthalmol: first published as 10.1136/bjo.76.4.210 on 1 April 1992. Downloaded from

Toshihiko Matsuo, Max S Cynader

Abstract in vitro binding technique and auto- F2a reduces intraocular pressure radiography; possibly by increasing uveoscleral outflow. To further understand the mechanism ofits action binding sites for prostaglandin F2a and, for Materials and methods comparison, were localised in sections of human cadaveric eyes using an in PREPARATION OF HUMAN EYE SECTIONS vitro ligand-binding technique and auto- Human cadaveric eyes were obtained within 24 radiography. Specific binding sites for both hours after death from the Eye Bank of British prostaglandin F2a and E2 were co-localised at a Columbia. The eyes used in this study had no high level in the areas ofthe ciliary muscles and documented ocular diseases. They were frozen iris sphincter muscles, and at a lower level in in isopentane cooled to -80°C and stored at the iris epithelium and the retina. The results -20°C until use. Sections of 20 iim thickness suggest that prostaglandin F2a and also prosta- were cutwitha cryostat (CambridgeInstruments, glandin E2, could modulate uveoscleral out- Nussloch, Germany) and placed on glass slides flow by binding to theirreceptors located on the coated with 1-7% gelatin. ciliary muscles and inducing their relaxation.

IN VITRO LIGAND BINDING AND AUTORADIOGRAPHY Prostaglandin F2,, and especially its more lipo- The sections were thawed at room temperature philic prodrug prostaglandin F2b-l-isopro- and preincubated in 50 mM tris-hydrochloride pylester can reduce intraocular pressure (IOP) buffer (pH 7 4) containing 100 mM sodium when applied topically to normal human volun- chloride, 3 mM calcium chloride, and 5% teers'23 and patients with glaucoma.45 Prosta- (weight/volume) bovine serum albumin (BSA, glandin F2a isopropylester is a very promising Sigma, St Louis, MO, USA) for 60 minutes at drug for glaucoma treatment because it has a room temperature. The sections were then in- prolonged potent hypotensive effect in low doses cubated in the same buffer containing 10-20 nM and lacks serious subjective or objective side of tritiated prostaglandin F2c, or E2 (Du Pont http://bjo.bmj.com/ effects. Canada, Mississauga, Ontario) for 90 minutes at A working classification of room temperature, washed for 60 minutes in the receptors for five naturally occurring prosta- ice-cold buffer containing 1% BSA, and finally glandins has been proposed based on pharma- dried in an air stream. The sections were then cological studies in which the agonist potencies apposed to tritium-sensitive films (Hyper- of naturally occurring as well as film-3H: Amersham Canada, Oakville, Ontario)

other synthetic agonists and antagonists were for 8 weeks in the dark. Non-specific binding on September 26, 2021 by guest. Protected copyright. compared.6 7 Receptors sensitive to was determined by incubating sections in the A2, , E2, F2,,, and I2 have been buffer containing, in addition, 100 ,uM un- designated as TP, DP, EP, FP, and IP-receptors, labelled prostaglandin F2c, or prostaglandin E2 respectively. The EP can be further (Cayman Chemicals, Ann Arbor, MI, USA) as classified into three subtypes, called EPI, EP2, well as 10-20 nM ofa labelled ligand. and EP3 receptors.89 The EP3 receptor has been shown to be involved in reduction of IOP in rabbits."'0 However it is not known which class Results ofprostanoid receptors is involved in the pressure Specific binding sites for both prostaglandin F2a reduction for humans and where within human and E2 were co-localised in the areas ofthe ciliary eyes the receptors for prostaglandin F2,, are muscles and iris sphincter muscles at a high located. level, and also in the iris epithelium and the There is evidence that prostaglandin F2a retina at a lower level as shown in Figures 1 and Department of lowers IOP by increasing uveoscleral outflow in 2. The lens structure showed apparently Ophthalmology, The University ofBritish monkeys.'2-'4 In order to further understand the moderate levels of the binding for both prosta- Columbia, Vancouver, basic mechanism of its action it is crucial to glandins, which were not distinctive compared Canada reveal the localisation ofbinding sites (receptors) with levels of the corresponding non-specific T Matsuo M S Cynader for prostaglandin F2a in the eye. It is also binding. The results were reproducible in three use eyes are Correspondence to: important to human since there eyes from three different individuals: a 60-year- Toshihiko Matsuo, MD, considerable anatomical and physiological old man who died of cerebral infarction, a 70- Department of Ophthalmology, Okayama differences between the eyes of different species, year-old woman who died of myocardial infarc- University Medical School, especially in the systems controlling aqueous tion, and a 52-year-old woman who died ofcolon 2-5-1 Shikata-cho, Okayama City, Okayama 700, Japan. humour outflow.'5 In this study we localised cancer. Accepted for publication binding sites for prostaglandin F2,, and also for The binding of 20 nM tritiated prostaglandin 6 September 1991 prostaglandin E2 in human eye sections using an F2a in all ofthe areas could be totally displaced by Localis,ation ofprostaglandin F2,, andE2 bindingsites in the human eye21211

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Figure I Autoradiographic viewzs ofprostaglandin F20,, binding sites in eye sections from a 60-year-old man. The binding of 20 nM tritiated prostaglandin F20 (A) concentrates in the ciliary muscle, iris sphincter muscle, iris epithelium, and the retina (and the detached retina). The binding was completely displaced by 100 pMl unlabelled (B) and by 100 p.M unlabelled prostaglandin F20,, (D). A Nissl-stained viewv ofA is given in C. http://bjo.bmj.com/

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Fig2B Fig2D Figure 2 Autoradiographic views ofprostaglandin H2 binding sites in eye sectionsfrom a 60-year-old man. The binding of 20 nM tritiatedprostaglandin B2 (A) concentrates in the ciliary muscle, iris sphincter muscle, iris epithelium, and the retina (and the detached retina). The binding was completely displaced by100p.M unlabelledprostaglandinEH2(B), andpartially displaced by 100 p.M unlabelledprostagladin F20,. (D). A Nissi stained viewz ofA is given inC. 212 Matsuo, Cynader

100 FM unlabelled prostaglandin F2a and induced by prostaglandin F2a in human eyes. prostaglandin E2 as shown in Figure 1. The The present experiments suggest that the ciliary

binding of 20 nM tritiated prostaglandin E2 muscles in human eyes have both EP receptors Br J Ophthalmol: first published as 10.1136/bjo.76.4.210 on 1 April 1992. Downloaded from could be totally displaced by 100 FtM unlabelled and FP receptors. Further studies using more prostaglandin E2, and partially and evenly in specific and sensitive ligands not presently avail- every area by 100 FtM prostaglandin F2a as able are required to answer the question ofwhich shown in Figure 2. specific class of prostanoid receptors plays the Retinal detachment commonly resulted during most important role in ocular hypotension. the processing of the eyes of autoradiography. Prostaglandin F2a did not change the produc- This separated the retina from other layers in the tion of aqueous humour by the ciliary epithelial posterior segment and made it clear that specific cells,'7 18 nor did it facilitate conventional outflow binding sites for prostaglandin F2a and E2 in the through the trabecular meshwork in humans4 7 posterior segment were located mainly in the or monkeys.'9 Instead prostaglandin F2, retina with minimal density in the choroid and apparently enhanced uveoscleral outflow in retinal pigment epithelial cells. The resolution of monkeys'2 13 and its pressure-lowering effect was the autoradiographic films was insufficient to antagonised by pilocarpine.'4 However the exact determine which specific layer in the retina, if mechanism by which it modulates the any, showed a high concentration of prosta- uveoscleral outflow remains unknown. glandin binding sites. Uveoscleral outflow can be affected by the tone ofthe ciliary muscles: contraction of the muscles induced by pilocarpine reduces the uveoscleral Discussion flow whereas their relaxation by atropine Prostaglandins are known to bind with pigments enhances the flow.2021 The binding sites for in the eye tissues.'6 In the present experiments prostaglandin F2CT appeared to be localised along the binding of prostaglandins to pigments could bundles of the ciliary muscle in the ciliary body. be reduced to less than 10% of the total binding It is therefore most probable that receptors for by preincubating and incubating the sections prostaglandin F2, are located on the ciliary with high concentrations (5%) of BSA and muscles. washing the sections with 1% BSA for durations Repeated doses ofprostaglandin F2a produced as long as 60 minutes. The conditions used are narrowing ofthe ciliary muscle fibres and widen- based on the fact that prostaglandins are lipo- ing of the intermuscular spaces in cynomolgus philic and bind to the pigments with a weaker monkeys22 and might also cause lysis and loss of affinity than they do to their receptors. intermuscular connective tissue.23 Its administra- In the present experiments, binding sites for tion was accompanied by partial reversal of prostaglandin F2a and prostaglandin E2 were co- resting myopia in monkeys24 but it did not induce localised to the same areas of the eye, primarily any measurable refractive changes in humans.3 4 the ciliary muscles and iris sphincter muscles, Although the facts mentioned above suggest

the iris epithelium, and the retina. The results that prostaglandin F2a could induce relaxation of http://bjo.bmj.com/ indicate that these areas had a mixture ofbinding the ciliary muscles there has been no evidence as sites (receptors) for both prostaglandins. How- to how prostaglandins would act directly on ever both prostaglandins can bind to the EP and ciliary muscles, whether they induce contraction FP prostanoid receptors at the ligand concentra- or relaxation of the muscles, or whether they act tions used in the experiments."' Further reduc- in a completely different way. Recently, prosta- tion of the ligand concentration to try to elicit glandin E2 (most potent), D2 and F2a, (less

more specific binding led to an inability to potent) were reported to induce relaxation of the on September 26, 2021 by guest. Protected copyright. visualise any binding sites. This failure is due to isolated longitudinal ciliary muscles of cats the limited resolution of the autoradiographic which had been contracted beforehand with techniques and to a relatively high noise/signal carbachol.2' The autoradiographic techniques ratio caused by the high 'background' binding of used here also demonstrated that the ciliary prostaglandins to the ocular pigments as muscle in cats had binding sites for prosta- mentioned above. glandin F2 and E2 as did in humans (data not The binding of prostaglandin F2a was totally shown). It will be necessary to examine further displaced by prostaglandin E2, whereas the bind- the direct effect of prostaglandins on the ciliary ing of prostaglandin E2 was not totally displaced muscle, especially in humans, to fully solve these by prostaglandin F2a. These results indicated questions. that most of the binding sites were receptive to Prostaglandin E2 and F2a can be synthesised both prostaglandin F2a and E2 at the 20 nM by cells lining the trabecular meshwork in concentrations, whereas there were some bind- humans.2627 These prostaglandins would remain ing sites receptive only to prostaglandin E2 at activefora while until theyflow orare transported that concentration. Thus the binding sites out of the eye2829 since ocular tissues in humans detected in this study would be a mixture of so far examined apparently do not contain heterogeneous types (EP receptors and FP which degrade prostaglandins meta- receptors) and subtypes (those of EP receptors) bolicaily.2" The present results support the idea ofthe prostanoid receptors. that prostaglandins synthesised in the trabecular The reduction of IOP by prostaglandins has meshwork would flow posteriorly in the aqueous been previously demonstrated to be mediated by humour and reach the ciliary body where they stimulation of the EP3 prostanoid receptor but may modulate uveoscleral outflow consequent to not of the FP receptor in rabbits.'0" It is still binding to their receptors located on the ciliary unknown which specific class of prostanoid muscle. Of course there is another possibility, receptors is involved in the pressure reduction namely, that the ciliary muscle itself might Localisation ofprostaglandinF2(, andE2 bindingsites in the human eye 213

under certain con- 10 Woodward DF, Burke JA, Williams LS, Palmer BP, Wheeler synthesise prostaglandins LA, Woldemussie F, et al. effects on ditions and thus might, in effect, turn on the intraocular pressure negatively correlate with FP-receptor stimulation. Invest Ophthalmol VisSci 1989; 30: 1838-42. uveoscleral outflow pathway. 11 Waterbury LD, Eglen RM, Faurot GF, Cooper GF. EP3, but Br J Ophthalmol: first published as 10.1136/bjo.76.4.210 on 1 April 1992. Downloaded from The fact that the iris sphincter muscles had not EP2, FP, or TP prostanoid-receptor stimulation may for F2a and E2 reduce intraocular pressure. Invest Ophthalmol Vis Sci 1990; binding sites prostaglandin 31: 2560-7. suggests that both prostaglandins might induce 12 Gabelt BT, Kaufman PL. Prostaglandin F2alpha increases miosis caused by their topical uveoscleral outflow in the cynomolgus monkey. Exp Eye Res miosis although 1989; 49: 389-402. administration has not yet been confirmed 13 Nilsson SFE, Samuelsson M, Bill A, Stiernschantz J; In- creased uveoscleral outflow as a possible mechanism of clinically.3' ocular hypotension caused by prostaglandin F2alpha-1- Prostaglandins play a role in the development isopropylester in the cynomolgus monkey. Exp Eye Res oedema after 1989; 48: 707-16. of cystoid macular especially 14 Crawford K, Kaufman PL. Pilocarpine antagonizes prosta- cataract extraction.32 Binding sites for prosta- glandin F2alpha-induced ocular hypotension in monkeys. found in the retina might Evidence for enhancement of uveoscleral outflow by prosta- glandin F2a, and E2 glandin F2alpha. Arch Ophihalmol 1987; 105: 1112-6. support the role of these compounds in cystoid 15 Bito LZ. Species differences in the responses of the eye to are known to be irritation and trauma: a hypothesis of divergence in ocular macular oedema. Prostaglandins defense mechanisms, and the choice ofexperimental animals actively transported out of the intraocular fluids for eye research. Exp Eye Res 1984; 39: 807-29. an acid transport system located in the 16 Aula P, Kaila T, Huupponen R, Salminen L, lisalo E. by organic Prostaglandin F2alphabindingtobovineocularand synthetic ciliary epithelium and retinal capillaries.2829 melanins in vitro. Pharmacol Toxicol 1989; 65: 100-3. there is a possibility that some of the 17 Kerstetter JR, Brubaker RF, Wilson SE, Kullerstrand LJ. Therefore Prostaglandin F2alpha-1-isopropylester lowers intraocular binding sites detected in the retina by the ligand- pressure without decreasing aqueous humor flow. Am would be the sites of this J Ophthalmol 1988; 105: 30-4. binding method 18 Camras CB, Podos SM, Rosenthal JS, Lee PY, Severin CH. transporter. This result also suggests a pos- Multiple dosing of prostaglandin F2alpha or epinephrine on that and E2 could play cynomolgus monkey eyes. 1. Aqueous humor dynamics. sibility prostaglandin F20 Invest Ophthalmol VisSci 1987; 28: 463-9. roles as retinal neuromodulators as has been 19 Gabelt BT, Kaufman PL. The effect of prostaglandin F2alpha in the on trabecular outflow facility in cynomolgus monkeys. Exp shown brain.33 Eye Res 1990; 51: 87-91. Receptor proteins for prostaglandin E2 and 20 Bill A. Uveoscleral drainage ofaqueous humor in human eyes. have been and shown to be ExpEye Res 1971; 12: 275-81. F20 solubilised 21 Bill A. Effects of atropine and pilocarpine on aqueous humor coupled with guanine nucleotide binding dynamics in cynomolgus monkeys (Macaca irus). Exp Eye proteins (G proteins).34 Recently a gene for the Res 1967; 6: 120-5. 22 Luetien-Drecoll E, Tamm E. Morphological study of the human receptor has been anterior segment of cynomolgus monkey eyes following cloned and shown to belong to the superfamily of treatment with prostaglandin F2alpha. Exp Eye Res 1988; 47:761-9. G protein-coupled receptor proteins. Expression 23 TammE,Luetien-DrecollE,RittigM,Rohen,JW. Connective of this cloned for thromboxane A2 revealed tissue changes in the uveoscleral pathways of primate eyes gene aftertreatmentwith prostaglandin F2alpha. ARVOabstracts. that the receptor responded only to thromboxane Supplement to Invest Ophthalmol Vis Sci. Philadelphia: A2 agonists but not to prostaglandin D2 or F20, Lippincott, 1989: 99. 24 Crawford KS, Kaufmann PL. Dose-related effects of prosta- supporting the working hypothesis that there glandin F2alpha isopropylester on intraocular pressure, might be a separate receptor for each of the refraction, andpupildiameterinmonkeys. InvestOphthalmol VisSci 1991; 32: 510-9.

naturally occurring . In the future 25 Chen J, Protzman CE, Woodward DF. Comparison of the http://bjo.bmj.com/ molecular biological approaches will further pharmacology of prostanoid induced ciliary muscle relaxa- tion and ocular hypotension reveals a correlation. ARVO enhance our understanding of the action of abstracts. Supplement to Invest Ophthalmol Vis Sci. Phila- prostaglandins in ocular tissues. delphia: Lippincott, 1991: 870. 26 Weinreb RN, Mitchell MD, Polansky JR. Prostaglandin production by human trabecular cells: in vitro inhibition by This work was supported by a MRC fellowship to TM and by dexamethasone. Invest Ophthalmol VisSci 1983; 24: 1541-5. from Pharmaceutical Co Ltd, Irvine, CA, USA. 27 Weinreb RN, Polansky JR, Alvarado JA, Mitchell MD. grants Allergan metabolism in human trabecular mesh- work cells. Invest Ophthalmol Vis Sci 1988; 29: 1708-12. 1 Giuffre G. The effects of prostaglandin F2alpha in the human 28 Bito LZ, Wallenstein MC. Transport of prostaglandins across on September 26, 2021 by guest. Protected copyright. eye. Graefes Arch Clin Exp Ophthalmol 1985; 222: 139-41. the blood-brain and blood-aqueous barriers and the physio- 2 Lee PY, Shao H, Xu L, Qu CK. The effect of prostaglandin logical significance of these absorptive transport processes. F2alpha on intraocular pressure in normotensive human Exp Eye Res 1977; 25 (suppl): 229-43. subjects. Invest Ophthalmol Vis Sci 1988; 29: 1474-7. 29 Bito LZ. Prostaglandins and other : their ocular 3 Villumsen J, Alm A. Prostaglandin F2alpha-isopropylester transport, , and therapeutic effects. Trans eye drops: effects in normal human eyes. BrJ Ophthalmol Ophthalmol Soc UK 1986; 105: 162-70. 1989; 73:419-26. 30 Cheng-Bennett A, Poyer J, Weinkam RJ, Woodward DF. 4 Camras CB, Siebold EC, Lustgarten JS, et al. Maintained Lack ofprostaglandin F2alpha metabolism by human ocular reduction ofintraocular pressure by prostaglandin F2alpha- tissues. Invest Ophthalmol Vis Sci 1990; 31: 1389-93. 1-isopropyl ester applied in multiple doses in ocular hyper- 31 Miranda OC, Bito LZ. The putative and demonstrated tensive and glaucoma patients. Ophthalmology 1989; %: miotic effects of prostaglandins in mammals. In: Bito LZ, 1329-37. Stiernschantz J, eds. The ocular effects ofprostaglandins and 5 Villumsen J, Alm A, Soederstroem M. Prostaglandin F2alpha- other eicosanoids. New York: Liss, 1989: 171-95. isopropylester eye drops: effect on intraocular pressure in 32 Miyake K. Prevention of cystoid macular edema after lens open-angle glaucoma. BrJ3 Ophthalmol 1989; 73: 975-9. extraction by topical indomethacin. I. A preliminary report. 6 Kennedy I, Coleman RA, Humphrey PPA, Levy GP, Lumley GraefesArch ClinExp Ophthalmol 1977; 203: 81-8. P. Studies on the characterization ofprostanoid receptors: a 33 Watanabe Y, Watanabe Y, Hamada K, Bommelaor-Bayt MC, proposed classification. Prostaglandins 1989; 24: 667-89. Dray F, Kaneko T, et al. Distinct localization of prosta- 7 Coleman RA, Humphrey PPA, Kennedy I, Lumley P. glandin D2, E2 and F2alpha binding sites in monkey brain. Prostanoid receptors. The development of a working classi- Brain Res 1989; 478: 143-8. fication. Trends Pharmacol Sci 1984; 5: 303-6. 34 Halushka PV, Mais DE, Mayeux PR, Morinelli TA. Throm- 8 Dong YJ, Jones RL, Wilson NH. receptor boxane, prostaglandin and receptors. Annu Rev subtypes in smooth muscle: agonist activities of stable Pharmacol Toxicol 1989; 10: 213-39. analogues. BrJ Pharmacol 1986; 87: 97-107. 35 Hirata M, Hayashi Y, Ushikubi F, Yokota Y, Kageyama R, 9 Eglen RM, Whiting RL. Characterization of the prostanoid Nakanishi S, et al. Cloning and expression of cDNA for a receptor profile of and isomers in smooth muscle human thromboxane A2 receptor. Nature 1991; 349: and in vitro. BrJ Pharmacol 1989; 98: 1335-43. 617-20.