110 BritishJournalofOphthalmology 1993; 77: 110-114 The EP2 receptor is the predominant prostanoid receptor in the human ciliary muscle Br J Ophthalmol: first published as 10.1136/bjo.77.2.110 on 1 February 1993. Downloaded from Toshihiko Matsuo, Max S Cynader Abstract IP prostanoid receptors, respectively. The EP Prostaglandins canreduce intraocularpressure receptor can be further classified into three by increasing uveoscleral outflow. We have subtypes, called EPI, EP2, and EP3 previously demonstrated that the human receptors.'89 The framework of the receptor ciliary muscle was a zone of concentration for classification has been supported in part, by binding sites (receptors) for prostaglandin F2a cloning and expression of cDNA for a human and for prostaglandin E2. Here, we try to thromboxane A2 receptor.20 elucidate the types of prostanoid receptors in It is important to know the types ofprostanoid the ciliary muscle using competitive ligand receptors located on the human ciliary muscle in binding studies in human eye sections and order to understand its role in uveoscleral out- computer assisted autoradiographic densito- flow, and to design new drugs with more potency metry. Saturation binding curves showed that and fewer adverse effects. In this study we tried the human ciliary muscle had a large number of to elucidate the type(s) of prostanoid receptors binding sites with a high affinity for prosta- located on the human ciliary muscle by glandin E2 compared with prostaglandin D2 combining receptor autoradiography with and F2,. The binding oftritiated prostaglandin competitive binding studies with various ligands E2 and F2a in the ciliary muscle was displaced on human eye sections. most effectively by prostaglandin E2 and 11- deoxy prostaglandin E1 (a selective EP2 prostanoid receptor agonist), whereas the Materials and methods binding of prostaglandin D2 was displaced most effectively by prostaglandin E2 and D2. PREPARATION OF HUMAN EYE SECTIONS These results indicate that the dominant Human cadaveric eyes were obtained within 24 prostanoid receptor in the human ciliary hours after death from the Eye Bank of British muscle is the EP2 subclass and that there is also Columbia. The eyes from two different a small number ofDP receptors. individuals were used: a 45-year-old man who (BrJ Ophthalmol 1993; 77: 110-114) died of heart failure and a 52-year-old woman who died of colon cancer. The eyes used in this study had no documented ocular diseases. They http://bjo.bmj.com/ Prostaglandins are known to reduce intraocular were frozen in isopentane cooled to -80°C and pressure in various species including rabbits, stored at -20°C until use. Sections of 20 tim cats, monkeys, andhumans, whengiventopically thickness were cut with a cryostat (Cambridge at low doses. The ability of prostaglandin F2a to Instruments, Nussloch, Germany) and placed on lower intraocular pressure has been well glass slides coated with 1-7% gelatin. documented in normal subjects'"3 and also in patients with glaucoma.4'5 The reduction of on October 1, 2021 by guest. Protected copyright. intraocular pressure following prostaglandin E2 IN VITRO LIGAND BINDING AND administration has been observed in cats and AUTORADIOGRAPHY monkeys," and the pressure lowering effect of The sections were thawed at room temperature prostaglandin D2 has also been examined in cats and preincubated in 50 mM TRIS-hydro- and rabbits." However, their effects in humans chloride buffer (pH 7-4) containing 100 mM have not been studied. sodium chloride, 3 mM calcium chloride, and There are several lines of evidence indicating 5% (weight/volume) bovine serum albumin that prostaglandin- F2,, lowers intraocular (Sigma, St Louis, MO) for 60 minutes at room pressure by increasing uveoscleral outflow in temperature. The sections were then incubated monkeys.'2-'4 We have previously demonstrated, in the same buffer containing a given concen- using an in vitro radioligand binding technique tration of tritiated prostaglandin D2, E2, or F2, Department of and autoradiography, that human ciliary muscle (Du Pont Canada, Mississauga, Ontario) for 90 Ophthalmology, The is a zone of concentration of binding sites University ofBritish minutes at room temperature, washed for 40 Columbia, Vancouver, (receptors) for prostaglandin F2a and for prosta- minutes in the ice-cold buffer containing 1% Canada glandin E2." bovine serum albumin, and finally dried in an air T Matsuo A working M S Cynader classification of prostanoid stream. The sections were then apposed to receptors for five naturally occurring prosta- tritium-sensitive Correspondence to: films (Hyperfilm-3H: Dr Toshihiko Matsuo, glandins has been proposed based on pharmaco- Amersham Canada, Oakville, Ontario) for 8 Department of logical studies in which the agonist potencies of Ophthalmology, The weeks in the dark. University of British naturally occurring prostaglandins as well as Columbia, 2550 Willow Street, other synthetic agonists and antagonists were Vancouver, BC, V5Z 3N9, Canada. compared.6 17 Receptors sensitive to throm- SATURATION AND COMPETITION BINDING STUDIES Accepted for publication boxane A2, prostaglandin D2, E2, F2a, and Sections were incubated with 1, 2, 5, 10, or 20 17 September. 1992 I2 have been designated as TP, DP, EP, FP, and nM concentration of tritiated prostaglandin Dz, The EP2 receptor is thepredominantprostanoid receptor in the human ciliary muscle III E2, or F2b to obtain a saturation binding curve for incubated with 10 nM of tritiated prostaglandin each prostaglandin. Specific binding at each D2, E2, or F2L, together with varying concen- concentration was obtained by subtracting non- trations (10' to 10' M) of seven different specific binding (determined by addition of 10 unlabelled ligands including prostaglandin D2, Br J Ophthalmol: first published as 10.1136/bjo.77.2.110 on 1 February 1993. Downloaded from FtM corresponding unlabelled ligand) from total E2, F2a, U-46619, 1 I-deoxyprostaglandinE1, 17- binding (determined without the unlabelled phenyltrinor prostaglandin F2a (all from Cayman ligand). The density ofbinding sites in the ciliary Chemicals), or sulprostone (Schering AG, muscle under different conditions was measured Berlin, Germany). The degree ofcompetition for as described below. each ligand was expressed as the relative percent- For competitive binding, sections were age between the binding of tritiated prosta- Fig IA Fig 11) http://bjo.bmj.com/ I Fig lB Fig IE on October 1, 2021 by guest. Protected copyright. Ql * t Wdi .... E ¢ , * , .' ,' k 0 1 -> _ - 9 PCig. IC(s ig IFI-, Figure 1 Autoradiographic views ofspecific binding sitesfor 2 nM tritiated prostaglandin D2 (A), E2 (B), and F2,, (C) in sections ofthe human eye...The .....binding , .sites ..for ..the # three prostaglandins are co-localised with concentrations in the ciliary muscle, iris sphincter muscle, iris epithelium, and retina. Non-specific bindingfor each prostaglandin determined by addition ofthe corresponding 10 MM unlabelled ligand is quite low. Nissl stained views ofthe same sections..for the specific :binding (A, .B, and ,C) are given tin D, E, and F, respectively. 112 Matsuo, Cynader -40 4) Br J Ophthalmol: first published as 10.1136/bjo.77.2.110 on 1 February 1993. Downloaded from 30 .-40E E 20 0C ._C Cu U)X3 10 -8 -7 -5 -4 --3 0)0 -9 -6 10~0 Log drug concentration (M) Figure 3 Binding curvesfor 10 nM tritiatedprostaglandin E2 in the human ciliary muscle after competition with varying concentrations ofunlabelledprostaglandin D2 (0), E2 (0), F2a (a), U46619 (I), Il-deoxy prostaglandin El (A), [3H]Prostaglandin concentration (nM) 17-phenyltrinorprostaglandin F2a (*), and sulprostone (A). Each value represents an average offour different 20 measurements. C co-exposed 3H-plastic standards ([3H] Micro- X 15 scales, Amersham). This enabled us to compare U) Figure 2 Saturation ._- the density of images from different films and binding curves (upperfigure) 0) 0 also to convert Gray scales to values of radio- and Scatchard plots (lower E activity per mg tissue. The density values in four figure)for specific binding of E 10 prostaglandin D2 (0), E2 different ciliary muscular areas from two eye (0), and F2,(E) in the sections studied under the same experimental Each L- human ciliary muscle. *0'a) conditions were averaged, as any one eye section value represents an average offour different c5 contained two areas ofthe ciliary muscle (see Fig measurements tn two eye 0 sections. The dissociation co constant (Kd) and maximum number ofbinding sites 0 Results (B..)for each 10 20 30 40 Figure shows autoradiographic views ofspecific prostaglandin are 1 summarised in Table 1. Prosttaglandin bound (fmol/mg tissue) binding sites for 2 nM concentrations of prosta- glandin D2, E2, and F2o in the human eye glandins obtained with a given concentration of sections. Specific binding sites for prostaglandin each competiLtive ligand and the total binding D2, E2, and F2a were concentrated in the same obtained without the competitive ligand. ocular tissues, namely, the ciliary muscle, iris http://bjo.bmj.com/ Competitive binding curves for one of the sphincter muscle, iris epithelium, and retina. unlabelled ligands versus the three tritiated The binding of 2 nM concentrations of each prostaglandinLs (prostaglandin D2, E2, and F2a) tritiated prostaglandin was displaced completely were drawn biy plotting relative autoradiographic by 10 FiM concentrations of the corresponding density values (%) in the ciliary muscle against unlabelled prostaglandin. concentrationLs