Adrenergic Receptors in Human Iris-Ciliary Body
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Distribution and Properties of /?-Adrenergic Receptors in Human Iris-Ciliary Body Martin D. Wax and Perry B. Molinoff 0-Adrenergic receptors from the iris-ciliary body of human eyes removed shortly after death were studied using membranes prepared by isopycnic centrifugation of tissue homogenates. This procedure separates uveal melanin pigment from plasma membranes and reduces nonspecific binding of I25I- iodopindolol. The observed binding of l25I-iodopindolol was of high affinity (K^ = 78 ± 6.6 pM), saturable, and fully reversible (U/2 = 4.6 min). Scatchard plots were linear and revealed a Bmax of 134 ± 20 fmol/ mg of protein from the whole iris-ciliary body. The affinities of the receptors for a series of agonists and antagonists were determined. The order of potency for the inhibition of the binding of the radioligand by antagonists was ICI 118,551 > MK950 > propranolol > ICI 89,406 > metoprolol. This order of potency is characteristic of /?-adrenergic receptors of the & subtype. Preparations of iris-ciliary body were also subjected to microdissection prior to density gradient centrifugation to permit the study of 0- adrenergic receptors in the ciliary processes, ciliary body, and iris. Each of these regions was found to contain approximately one third of the total number of /?-adrenergic receptors in the human iris-ciliary body. The highest density of receptors was located in the ciliary processes (180 ± 40 fmol/mg of protein), while the density of receptors in the iris (98 ± 7.5 fmol/mg of protein) and ciliary body (less the processes) (42 ±17 fmol/mg of protein) was notably lower. Only /?2-adrenergic receptors are detectable by competition experiments in the iris-ciliary body as a whole, or in the individual preparations of iris, ciliary processes, or ciliary body; however, microdissection and analysis of 0-adrenergic receptor subtypes in isolated ciliary muscle permitted detection of a small number of/8,-adrenergic receptors. /Si-Adrenergic receptors comprised about 10% of the total number of 0-adrenergic receptors in the whole iris-ciliary body. The finding that most of the /?-adrenergic receptors in the human iris-ciliary body are of the 02 subtype may be of significant therapeutic importance in the medical management of glaucoma. Invest Ophthalmol Vis Sci 28:420-430, 1987 Physiological and biochemical studies support the Binding assays with radioligands are widely used to importance of /3-adrenergic receptors in the regulation study /3-adrenergic receptors and receptor subtypes. The of intraocular pressure.1 The ciliary processes are basic technique involves monitoring inhibition of the known to be the active secretory site for the production binding of a radiolabeled compound, usually an an- of aqueous humor. In addition to /3-adrenergic recep- tagonist, by various concentrations of unlabeled ago- tors located on the secretory ciliary epithelium, there nists or antagonists. The identification and pharma- are receptors in the trabecular mesh work and paratra- cological characterization of /3-adrenergic receptors in becular regions. /3-Adrenergic receptors also exist in the human eye have been hampered by the presence the anterior segment microvascular bed from which of melanin pigment in the uvea, which interferes with aqueous humor ultrafiltrate is derived and into which the binding of radioligands. Melanin binds radioli- it is partially resorbed.2 The properties and functional gands, causing a marked increase in "nonspecific" role of the /3-adrenergic receptors in each of these areas binding (ie, ligand not bound specifically to the /3-ad- need to be denned for a full understanding of the mod- renergic receptor). This increases the noise of the assay ulation of aqueous humor dynamics by drugs or neu- such that the remaining specific binding cannot be re- rotransmitters. liably detected. Thus, prior investigations of /3-adren- ergic receptors in the anterior segment have utilized preparations from pigment-free specimens, such as the 3 5 From the Department of Pharmacology, University of Pennsyl- albino rabbit, " or preparations of isolated ciliary 6 8 vania School of Medicine, Philadelphia, Pennsylvania. processes " of pigmented species. Successful efforts to Supported by USPHS Grants NS18479, GM34781 and MH14654. remove melanin utilizing discontinuous sucrose gra- Submitted for publication: January 3, 1986. dients to detect /3-adrenergic receptors in the ciliary Reprint requests: Martin B. Wax, MD, Department of Pharma- processes of sheep have been described.8"10 cology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6084. A rapid, convenient method has been developed that 420 Downloaded from iovs.arvojournals.org on 09/27/2021 No. 3 0-ADRENERGIC RECEPTORS IN HUMAN IRIS-CILIARY BODY / Wax and Molinoff 421 utilizes isopycnic centrifugation to separate plasma pigment formed a globular pellet at the bottom of the membranes with /3-adrenergic receptors in human iris- tube at a density greater than 1.09 g/ml. The properties ciliary body preparations from melanin and cyto- of /?-adrenergic receptors were characterized on gra- plasmic contaminants. This procedure results in good dient fractions excluding the melanin-containing pellet. recovery of membrane-bound receptors and avoids All of the gradient above the melanin pellet was re- problems that result from the presence of melanin. The moved and the membranes contained in the Percoll results suggest that the /3-adrenergic receptors in mem- gradient were resuspended in 20 mM Hepes buffer brane homogenates of human iris-ciliary body are pre- containing 145 mM NaCl prior to their addition in the dominantly of the j82-adrenergic receptor subtype. The binding assays. Percoll remained in the continuous current medical management of glaucoma relies pri- gradient which formed after centrifugation, enabling marily on the topical ocular application of nonselective the use of membranes from various regions of the gra- antagonists such as timolol and agonists such as epi- dient in the subsequent binding assays. nephrine to lower intraocular pressure. The use of drugs 125 which act selectively at jSr or /32-adrenergic receptors Preparation of ( I)-Iodopindolol may offer distinct advantages in efficacy, in addition (125I)-iodopindolol (125I-IPIN) was prepared by a to minimizing undesirable systemic side effects, when 1 compared with the use of nonselective agents. modification of the method of Barovsky and Brooker ' as described by Wolfe and Harden.12 Ten n\ of ethyl acetate containing 13 ng of (—)-pindolol was placed in Materials and Methods a polypropylene tube, and the solvent removed by Tissue Preparation evaporation. Twenty ^1 of 0.3 M KH2PO4 (pH 7.4), 20 n\ of chloramine T (0.34 mg/1), and 20-50 »\ (2-5 Freshly enucleated eyes from human donors (ages mCi) of Nal25I were added, and the mixture was in- 14 to 91) were obtained within 24 hr of death (primarily cubated for 3 min at room temperature. The reaction due to cardio-respiratory failure) and were dissected was stopped by addition of 300 n\ of Na2S2O3 (1 mg/ on ice in 20 mM Hepes buffer (pH 7.5) containing 145 ml) and 10 n\ of 1 N NaOH. Iodinated product was mM NaCl. The pars plana was incised 4 mm posterior extracted 3 times into 300 n\ of ethyl acetate containing io me corneoscleral limbus and the anterior segment 0.01% phenol. The extracts were combined and spotted was removed intact from the scleral spur by surgical on 3M chromatography paper. Descending chroma- separation. Histopathologic confirmation of represen- tography was run for 4-6 hr at room temperature in tative cross sections assured the inclusion of all para- 0.5 M ammonium formate (pH 8.5) containing 10% trabecular tissue and iris root in the iris-ciliary body methanol and 0.01% phenol. The chromatogram was preparation, leaving only cornea and sclera in the dis- cut into strips and the ligand extracted into methanol carded remnant. Each iris-ciliary body was suspended containing 0.01% phenol. Typically, 60-80% of the I25I in 30 ml of 20 mM Hepes buffer containing 145 mM was incorporated into the radioligand. The ligand had NaCl, homogenized with a Brinkmann Polytron (5 a specific activity of 2200 Ci/mmol, was stored at times for 1 sec at setting 6-7), and centrifuged at 20,000 -20°C, and was utilized for up to 3 weeks. X g. Membrane preparations were either used directly or stored frozen at -70°C. Frozen membranes were thawed and homogenized in a Wheaton glass homog- Assay of 0-Adrenergic Receptors enizer fitted with a teflon pestle prior to use. The prop- Properties of /3-adrenergic receptors were assessed erties of the receptors were unaffected by storage of by adding 100 iA of a membrane suspension (5-10 ng homogenates for up to 3 months at —70°C. Percoll of protein) to a mixture containing 50 ^1 of (125I)-IPIN (Pharmacia Fina Chemicals, Uppsala, Sweden), a col- in bovine serum albumin (BSA) (10 ng/m\) and ascor- loid suspension of silica coated with poly vinyl pyrrol- bic acid (0.25 mg/ml), 50 /A of BSA/ascorbate or drug, idone, was diluted from stock to initial concentrations and 50 /d of 500 nM GTP in 20 mM Hepes buffer of 20% and 25% with 20 mM Hepes buffer containing containing 0.9% NaCl. Specific binding was defined as 145 mM NaCl. The pellet formed by centrifugation of the amount of (125I)-IPIN bound in the absence of a a homogenate of a single eye was resuspended by mix- competing ligand minus the amount bound in the ing it with a Percoll suspension in a Beckman Polyal- presence of 200 nM (-)-timolol maleate. Equilibrium lomer (Palo Alto, CA) centrifuge tube (16 X 76 mm). binding was performed for 20 min at 37°C in dispos- The suspension was centrifuged at 20,000 rpm in a able polypropylene tubes (Walter Sarstedt, Princeton, 2 9 2 70.1 Ti rotor (w t = 1.3-2.6 X 10 rad /sec) to form a NJ).