Investigative Ophthalmology & Visual Science, Vol. 32, No. 6, May 1991 Copyright © Association for Research in Vision and Ophthalmology Localization and Characterization of Substance P Binding Sites in Rat and Rabbit Eyes Philippe Denis,*t Veronique Fardin4 Jean-Philippe Nordmann,t Pierre-Paul Elena,§ Laurent Laroche,-(- Henri Saraux,t and William Rostene* Specific and high-affinity binding sites for Substance P (SP) were found in eyes from albino rabbits and rats using an in vitro autoradiographic method with l2SI-Bolton Hunter SP (BHSP). Autoradiograms were generated by apposing 10-20/im-thick cryostat eye sections to 3H-Hyperfilm or liquid emulsion and quantified by means of image-analysis procedures. Kinetic studies showed that equilibrium was reached after a 75-min incubation at room temperature. In rat retina, specific binding corresponding to approximately 90% of total binding, was reversible, of high affinity (dissociation constant [Kd], 0.13 ± 0.02 nM). Half-time for dissociation of 125I-BHSP was about 15 min. I) n la be led SP and the two neurokinins (NK) A and B competed in a concentration-dependent manner for retinal sites labeled by 125I-BHSP with the following order of potencies: SP > NKA > NKB, in agreement with a pharmaco- logic profile of a SP receptor site. In both species, specific binding was found in the iris sphincter muscle, choroid, and retina. In rats, detectable amounts of SP-binding sites were also expressed in the corneal epithelium and iridial stroma. Quantitative analysis of the autoradiograms revealed that the highest densities of 125I-BHSP binding sites were localized in the iris sphincter muscle in rabbits and the inner retina in rats. Invest Ophthalmol Vis Sci 32:1894-1902,1991 Numerous neurogenic mediators released in the an- such as calcitonin gene-related peptide (CGRP) or terior segment of the eye by ocular injury, trauma, or cholecystokinin, have also been identified in ocular noxious stimulation are known to elicit inflammatory sensory structures8'9 and shown to play a functional effects, such as conjunctival hyperemia, miosis, rise in role in the neurogenic inflammation (particularly intraocular pressure, and disruption of the blood- blood-aqueous barrier breakdown for CGRP.l0) How- aqueous barrier.' If some of these biologic effects are ever SP involvement in neurogenic inflammation is demonstrated to be mediated directly by metabolites suggested by several pieces of evidence. First, nerve from the arachidonic acid cascade (released from the endings with immunoreactivity to SP are found in the iris and the ciliary body),2 it is now widely recognized uvea of several species, including humans," mainly in that neural pathways also participate in the initiation close association with the sphincter muscle of the iris of such inflammatory events by releasing neuropep- and the smooth blood vasculature in the ciliary body. tides from sensory afferent nerves in the uveal tract.3 Second intracameral administration of SP induces a Substance P (SP), an undecapeptide isolated from in- dose-dependent, nonmuscarinic pupil constriction testine in 1931,4 was first proposed as a neurogenic associated with aqueous flare and an increase in intra- mediator of antidromic vasodilation and plasma ex- ocular pressure in the rabbit eye.6 Third electrical stim- travasation at the peripheral level5 and as a major ulation of the trigeminal ganglion or intracameral ad- component in the neurogenic ocular injury re- ministration of capsaicin (both responsible for SP re- sponses.6'7 Later, other biologically active substances, lease in the anterior segment) are able to mimic SP-induced miosis.61213 Fourth, (D-Pro2, D-Trp7-9)- SP, a SP antagonist, counteracts this phenomenon in rabbits and could therefore inhibit the ocular inflam- From the *1NSERM U55 and tDepartment of Ophthalmology, 14 Hopital Saint Antoine, Paris, JRhone-Poulenc Sante, Department matory response to laser iridial burns. of Biology, Vitry-Sur-Seine, and the §Department of Pharmacol- Biochemical and immunohistochemical studies ogy, Faculte de Medecine, Nice, France. have localized SP in various vertebrate retinas.15 Cel- Philippe Denis was a recipient of INSERM (Poste d'accueil). Submitted for publication: September 25, 1990; accepted . lular expression of SP-encoding mRNA was found re- Reprint requests: Philippe Denis, INSERM U55, 184, rue du cently in the rat retina using RNA blot and in situ 16 Faubourg Saint Antoine, 75012, Paris, France. hybridization. Although physiologic studies indi- 1894 Downloaded from iovs.arvojournals.org on 10/01/2021 No. 6 OCULAR SUBSTANCE P BINDING SITES / Denis er al 1895 cate that SP has a neuromodulator action on ganglion taining 10 mM MgCl2,2 g/1 of bovine serum albumin, cells in fish17 and in dopamine release from the retina 40 mg/1 of bacitracin, 5 mg/1 of leupeptin, and 4 mg/1 in the rat,18 the role of SP immunoreactive neurons in of bestatin. Nonspecific binding was determined on the processing of visual information is not yet fully alternate sections in the presence of 10 ju.M unlabeled understood. peptide (SP) added in the incubation medium. The The presence of ocular SP binding sites was sug- specificity of the binding was studied by incubating gested previously by conventional binding techniques sections with increasing concentrations of peptides using membranes obtained from rat and bovine ret- related to SP, such as senktide, septide, spantide, ina1920 or bovine and rabbit iris,2122 but the precise SP methylester, neurokinin A (NKA), neurokinin distribution of these receptor sites has not yet been B (NKB), and synthetic fragments of SP (SP,_4 and investigated extensively. One autoradiographic report SP4_n). Bacitracin, leupeptin, and bestatin were ob- briefly mentioned the presence of binding sites in the tained from Sigma (St. Louis, MO) and SP-related rat retina,23 but no quantitative data or information peptides from Bachem (Bubendorf, Switzerland). on the pharmacologic profile of SP binding were pre- After incubation with the radioligand, the sections sented. It is important to localize these binding sites were rinsed four times for 1 min each in the preincu- since it is believed that most SP biologic actions are bation buffer at 4°C, dipped for 20 sec into distilled receptor -mediated.24 We therefore characterized and water, and quickly dried using a stream of cold air for determined the anatomic localization of SP binding autoradiography. sites in rat and rabbit eyes using quantitative in vitro autoradiographic methods. We have used Bolton- Autoradiographic Experiments Hunter SP (125I-BHSP; Amersham, les Ulis, France), The sections were preincubated as described and a radiolabeled analogue of the tachykinin which has 125 been extensively used in other organs to label SP re- incubated with 65 pM I-BHSP for 90 min at room ceptors.25"27 temperature. After washing, the dry slides were stored in a Kodak X-ray cassette (Rochester, NY) in tight apposition to a tritium-sensitive film (3H-Hyperfilm; Materials and Methods Amersham) and allowed to expose for 1 week in dark- Tissue Preparation ness. After exposure, the films were developed in Ko- New Zealand albino rabbits (weighing 3-3.5 kg) dak D19 for 3 min and fixed. To identify the localiza- were killed by injection of a lethal dose of sodium tion of the binding sites, the eye sections were coun- pentobarbital and Wistar rats (weighing 200-250 g), terstained with hematoxylin and eosin and cover by decapitation. The eyes were removed, immersed in slipped with Fluka (Chemika, Buchs, Switzerland). Tissue Tek medium (Miles Scientific, Naperville, IL), To investigate precisely the cellular localization of frozen in isopentane cooled (-40°C) in liquid nitro- SP binding sites, an autoradiographic technique was gen, and stored at -80°C. Just before sectioning, the used at light microscopic resolution. Briefly, after in- tissues were warmed to -20°C, and sections (20-fxm cubation and washings, selected slides were treated thick) were cut with a cryostat (Bright), thaw- with a 30-min bath of 4% glutaraldehyde at 4°C to fix mounted onto gelatin-coated glass slides, and stored covalently the radioligand to its binding site, defatted at -80°C until use. Before incubations, sections were in several baths of increasing concentrations of alco- allowed to thaw at room temperature. All investiga- hol (75-100%) and xylene, and then dipped into liq- tions described in this paper were done in accordance uid nuclear emulsion (LM1,; Amersham). Prelimi- nary experiments showed that this treatment did not with the ARVO Resolution on the Use of Animals in 125 Research. significantly alter I-BHSP binding. After an expo- sure period of 10 days in darkness, the emulsion Binding Conditions dipped microautoradiograms were developed and fixed as described. Corresponding sections were coun- Preliminary experiments showed that preincuba- terstained and examined under a light microscope. tion was found to increase specific binding. Thus, Light- and dark-field photomicrographs were taken slides were washed in a preincubation medium (Tris from the stained sections and silver grains, respec- -HC1 50 mM, pH 7.4, containing 0.2 g/1 of bovine tively. serum albumin) at room temperature for 15 min be- fore incubation with radioligand. After the washing step, the slides were then incubated at room tempera- Data Analysis ture in a solution of 65 pM 125I-BHSP (2000 Ci/ Film macroautoradiograms were analyzed by com- mmol) [Bolton-Hunter is 3-(p-hydroxy-m-(125I)iodo- puter-based densitometry. The optical density of the phenyl)-propionyl) in 50 mM Tris -HC1, pH 7.4, con- autoradiogram was quantified by means of an image Downloaded from iovs.arvojournals.org on 10/01/2021 1896 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1991 Vol. 32 analyzer (BIOCOM RAG 200, les Ulis, France). Briefly, autoradiograms were digitized, and each grain density was assigned a relative optical density value. For maximal binding capacity determination, these relative optical density values were then converted to corresponding commercial 125I Amersham standards, and the results were expressed in fmol/mg tissue equivalent.