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 ligand binding technique and auto- Prostaglandin 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, prostaglandin E2 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 prostanoid 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 prostaglandins 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 thromboxane was determined by incubating sections in the A2, prostaglandin D2, 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 receptor 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 A Br J Ophthalmol: first published as 10.1136/bjo.76.4.210 on 1 April 1992. Downloaded from 'I. I - Pr' FiglIA FigIC N .:7 I Fig lB Fig ID 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 prostaglandin H2 (B) and by 100 p.M unlabelled prostaglandin F20,, (D). A Nissl-stained viewv ofA is given in C. http://bjo.bmj.com/ Wal I on September 26, 2021 by guest. Protected copyright. I Fig 2A Fig 2C 'I 7 .1 I .41 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.