Localization and Characterization of Substance P Binding Sites in Rat and Rabbit Eyes

Home , Iris sphincter muscle

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 pharmacologic 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 responses.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 exposure 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 before 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. Amersham's microscales are supplied as o slices of several layers of polymer containing a range 00 of increasing 1251 concentrations. Since tissue sec- 00 tions are coexposed with iodinated plastic standards, we can do quantitative densitometry using a computer-assisted system. Tissue-equivalent values were provided based on calibration using intact brain gray matter. In all cases, specific binding was defined by 11 10 9 8 7 6 subtracting nonspecific binding (obtained in the pres- SP concentration Log.M ence of 10 iiM of unlabeled SP) from total binding. 125 Classic computer analysis was used for the biochemi- Fig. 2. Competition between I-BHSP and unlabeled SP for binding to rat retina sections. 65 pM 125I-BHSP were incubated cal determination of the binding parameters (dissocia- alone or in the presence of increasing concentrations of unlabeled tion constant [Kd] and concentrations which inhibit peptide SP at room temperature for 90 min. 125I-BHSP binding is 50% of the specific binding [IC50]) on rat retina sec- expressed as a percent of total binding. Values are given as the mean tions. The kinetics of I25I-BHSP binding was assumed of triplicate determinations. to follow a bimolecular association model (second- order kinetics). BHSP resulted in a time-dependent increase in bind- Results ing (Fig. 1). At room temperature, specific binding Characterization of 125I-BHSP Binding reached a plateau in approximately 75 min; nonspe- Kinetic analysis done by densitometry on rat reti- cific binding was not significantly increased. About nal sections indicated that the association of 125I- 90% of total binding was specific at 75 min. A routine incubation time of 90 min was then adopted for all subsequent experiments. At equilibrium, bound 125I- 8O - BHSP could be dissociated specifically from its binding sites by incubating sections with an excess (10 ixM) of unlabeled peptide (Fig. 1). The half-time for dissociation of 125I-BHSP was about 15 min. We investigated competition studies between iodinated SP and increasing concentrations of unlabeled peptide (Fig. 2). These results indicated that SP inhibited 125I-BHSP competitively and with very high affinity. In the range of concentrations studied (10"u 6 to 10~ M), IC50 values were 0.17 nM. Curve analysis obtained (obtained by regression lines computerized by means of the least-squares method) indicated that 125I-BHSP bound to high-affinity binding sites in rat retina with an apparent Kd estimated at 0.13 5O TOO 15O ± 0.02 nM. Time(min) Competition studies with the tachykinins NKA, I25 NKB, and analogues or fragments of SP were done in Fig. 1. Association and dissociation of I-BHSP binding to rat I25 retina sections. Retina sections were incubated with 65 pM 125I- attempt to establish the type of site identified by I- BHSP at room temperature for various times. The curve represents BHSP in the rat retina. The order of potency for inhi- specific binding that was calculated as the difference between bind- bition of binding was SP > NKA > NKB (Fig. 3, Ta- ing in the presence and absence of 10-^M unlabeled substance P ble 1). A similar ranking was obtained when rabbit (SP). Dissociation was initiated by the addition of 10-juM unlabeled eye sections were incubated with the tachykinins at SP at 120 min, a time at which the association of the ligand had 8 5 reached the equilibrium. Values are given as the mean of triplicate concentrations in the range of 10" to 10~ M (data determinations in a typical experiment. not shown). Displacement curves showed that specific

Downloaded from iovs.arvojournals.org on 10/01/2021 No. 6 OCULAR 5UDSTANCE P BINDING SITES / Denis er al 1897

plexiform layer and proximal inner nuclear layer (INL). Much lower SP binding was present in other regions, particularly in the outer retina (outer nuclear .SP layer and photoreceptor cell layer). Similarly, very ASP4-11 • NKA low, but significant, concentrations of SP binding 0SPl-4 sites were seen in the choroidal tissue. Some scleral ANKB blood vessels also exhibited some binding activity (Fig. 5, see arrows). In the rabbit eye (Fig. 6), the topographic distribution of SP binding sites was similar to that found in the rat although there were some quantitative differences (Table 2). Very high labeling was seen in associa- 11 10 9 8 7 6 tion with the iridial sphincter muscle; no labeling was Peptide concentration -Log M found either in the cornea, iris stroma, or ciliary body. Fig. 3. Pharmacology of 125I-BHSP binding. 65 pM 125I-BHSP Low binding was also identified in the choroid. As in were incubated alone or in the presence of increasing concentra- the rat, lens fibers gave important nonspecific bind- tions of unlabeled peptides at room temperature for 90 min. I25I- ing, ie, not displaceable in the presence of an excess of BHSP binding is expressed as a percent of initial binding. Values are unlabeled peptide. The retina also had high densities given as the mean of four determinations. of specific SP binding sites, mainly concentrated in the IPL and GCL, as described in the rat. I25 binding of I-BHSP was strongly inhibited by Discussion SP,_n (IC50=, 0.17 ± 0.02) (Fig. 2) and to a lesser extent by SP4_n and NKA (IC50=, 3.39 and 8.04 nM, These results demonstrate that several structures respectively). On the other hand, senktide and septide from albino rat and rabbit eyes (mainly the retina and was ineffective, and SP^, NKB, and spantide had the iris sphincter muscle) have the capacity to bind some potency to inhibit retinal I25I-BHSP binding specifically a derivative analogue of SP, I25I-BHSP. (Table 1). The characteristics of I25I-BHSP binding in the rat retina strongly suggest that the ligand binds to SP re- Autoradiographic Experiments ceptors. The apparent Kd of the high-affinity (0.13 nM) Representative autoradiograms of 125I-BHSP bind- binding site identified in this study was similar to ing sites are shown in Figure 4-6. In the rat anterior 19 those reported in rat retina homogenates (0.2 nM), segment, SP binding sites were found in the iris, 28 in rabbit optic sections (0.54 nM), and in human mainly in the sphincter region (Fig. 4, Panel a). Analy- 29 retina (0.27 nM). Recent studies have described the sis of histologic sections confirmed that labeling on existence of multiple tachykinin receptors in the rat the pupil margin coincided with the iris muscular tis- 23 retina. The agent, SP, belongs to a group of closely sue. The density of silver grains was reduced dramati- cally in the presence of 10 IJM unlabeled SP (Fig. 4, Panel b), indicating specific labeling in that structure. Table 1. In contrast, moderate 125I-BHSP binding densities were noted over the other iridial structures and in the Peptides ICso (nM) corneal epithelium. No labeling occurred in struc- Substance P 0.17 ±0.02 tures such as the sclera and the ciliary processes. Bind- Substance P^,, 3.39 ± 0.40 ing seen on the lens fibers was not considered to be Neurokinin A 8.04 ± 0.57 specific since SP did not displace 125I-BHSP labeling. Substance P,_4 35 ±4.34 Neurokinin B 52.8 ± 4.06 In the posterior segment, the retina showed intensive Spantide 53 ± 1.53 labeling; low concentrations of binding were noted in Substance P methyl ester 54 ± 6.34 the choroid. Binding sites were not uniformly distrib- Septide >1000 uted throughout the retina since a slight decrease of Senktide >1000 binding at the ora serrata was observed. Autoradio- Displacement of 125I-BHSP binding on rat retina sections by substance P (SP), peptides related to SP, and synthetic fragments of SP. Sections were grams of the emulsion-dipped slides provided detailed incubated for 90 min at room temperature with 65 pM I251-BHSP and in- histologic resolution of the retinal SP binding sites creasing concentrations (10"" to 10~6 M) of unlabeled peptides. Nonspecific binding of the radioligand was determined in the presence of 10-MM unla- (Fig. 5). Silver grains were localized primarily in the beled SP. IC50 is defined as the molar concentration of the tested drug that inner plexiform (IPL) and ganglion cell layers (GCL). displaced 50% of the specific binding of I25I-BHSP on retinae sections. Autora- diograms were analyzed by computer-based densitometry with an image ana- A few scattered grains were also detected in the outer lyser. Values are given as the mean of triplicate determinations.

Downloaded from iovs.arvojournals.org on 10/01/2021 1898 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1991 Vol. 02

the mammalian tachykinins have been described, namely SP (or NK-1) receptors, found in both central and peripheral tissues, NKA (or NK-2) receptors in ISM peripheral tissues, and NKB (or NK-3) receptors mainly detectable in the central nervous system.23'32 Our data suggest that 125I-BHSP binds preferentially /|SM to NK-1 receptor sites; SP is the most potent tachykinin in inhibiting 25I-BHSP binding and the order of potency of the competitors for 125I-BHSP binding was SP > NKA > NKB. The IC50 values calculated for NKA and NKB were somewhat lower than those found for the displacement of I25I-BHSP binding in a preliminary report.19 Variations in the methods, including the incubation buffer and the lower ligand concentration used in our study (65 pM), may be the cause of these differences. It is unlikely that I25I-BHSP binds to NKB receptors since senktide, a highly selective NK-3 agonist, showed no activity at the micro- molar level in displacing 125I-BHSP from its binding

Fig. 4. Autoradiograms of I25I-BHSP binding in albino rat eye. Sections were incubated for 90 min at room temperature with 65 pM I251-BHSP alone (A) or in presence of 10-^M unlabeled SP to determine nonspecific binding (B). Autoradiograms were generated by apposition to 3H-Hyperfilm for 7 days. (C) Histological section, (c, cornea; ism, iris sphincter muscle; cp, ciliary processes; 1, lens; ch, choroid; r, retina). Magnification X3. B Fig. 5. Microautoradiograms of I25I-BHSP binding in albino rat posterior segment. Dark field (A) and bright field (B) were obtained related peptides known as tachykinins, all of which after incubation of 65 pM '"l-BHSP alone. Silver grains visualized contain a common C-terminal amino acid sequence: in the retina and choroid indicated specific SP binding sites since 30 labeling disappeared after incubation with an excess of unlabeled Phe-X-Gly-Leu-Met-NH2. In 1983, two additional peptide (not shown). GCL, ganglion cell layer; IPL, inner plexiform tachykinins, NKA and NKB, were isolated in mam- layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, malian tissues.31 To date, three distinct receptors for outer nuclear layer; SV, scleral vessel. Magnification X500.

Downloaded from iovs.arvojournals.org on 10/01/2021 No. 6 OCULAR SUBSTANCE P BINDING SITES / Denis er ol 1899

binding sites were found to be mainly associated with the iris sphincter muscle although other iridial structures were slightly labeled in the rat eye. The presence of SP was reported in sensory neurons of the anterior 3334 ISM uvea originating from the trigeminal ganglion, and a number of investigators extensively studied the involvement of the peptide in the acute irritation response of the eye. Besides its vasodilator properties, early experiments reported that the tachykinin peptide induces an increase in intraocular pressure and moderate alterations in the blood-aqueous barrier in rabbit eyes.6'714'35 Subsequent studies observed that these effects were relatively weak and inconsistent and that the rise in intraocular pressure was mainly due to a miosis-induced pupillary blockade since it was abolished in part by peripheral iridectomies.12 Despite its wide distribution in the uveal tract in many species,11'33'34'36'37 SP has been proposed to be only responsible for the miotic component of the antidromic ocular injury response.12'3538 The other parts of the inflammatory response (elevated intraocular pressure and blood-aqueous barrier breakdown) are thought to be mediated by other mechanisms; in animals pretreated with prostaglandin inhibitors, injection of SP caused only miosis.12 The release of CGRP B from sensory nerves has also been shown to induce marked inflammatory effects in cat39 and rabbit40 eyes. The two neuropeptides are partially colocalized in terminal endings arising from the trigeminal cells41 and are released together into aqueous humor during the antidromic response. In addition, SP-induced miosis is potentiated by CGRP,42 suggesting biologic interactions between the two peptides. Our autoradiographic results provide essential information con- cerning the role of SP in the ocular anterior segment; they demonstrate the presence of high-affinity binding sites for SP associated with the iridial sphincter muscle. They may also suggest that the miotic response to SP in rabbits may be due to a direct action

Fig. 6. Autoradiograms of I25I-BHSP binding in albino rabbit anterior segment. Sections were incubated for 90 min at room temperature with 65 pM I25I-BHSP alone (A) or in presence of 10-^M Table 2. unlabeled SP (B). Auloradiograms were generated by apposition to 3H-Hyperfilm for 7 days. (C) Histological section, C, cornea; ISM, Specific l2iI-BHSP bound iris sphincter muscle; CP, ciliary processes; L, lens. Magnification Area (fmol/mg tissue equivalent) X3, Cornea rat 0.12 ±0.01 Iris stroma rat 0.11 ±0.01 Iris sphincter muscle rabbit 2.93 ± 0.42 site. Furthermore, as described for SP binding sites in rat 0.17 ±0.01 l9 25 26 Retina rabbit 0.95 ±0.17 other tissues, ' > affinity is encoded in the carboxy rat 0.63 ± 0.07 terminal of SP since SP,_, j and SP4_n had the greatest potency in inhibiting I25I-BHSP binding. Regional distribution of specific BHSP binding sites in the albino rat and 125 rabbit eye. Results were obtained by transformation of optical densities as Autoradiographic visualization of I-BHSP bind- described in the text to fmol/mg tissue equivalent ± SEM and were obtained from at least 10 determinations throughout the area under examination. ing sites in the anterior segment of the eye shows a Nonspecific binding was subtracted from each total value, so that data are selective pattern of distribution. In both species, SP expressed as specific binding.

Downloaded from iovs.arvojournals.org on 10/01/2021 1900 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1991 Vol. 32

on the iris muscle through specific receptors. No bind- tal conditions, competition studies with increasing ing sites was found in dilator or ciliary muscles, cor- concentrations of tuftsin (10~4 to 10~7 M) showed that roborating the fact that SP has no contractile effect on 125I-BHSP binding was not inhibited by tuftsin (data these tissues.3843 One of the most interesting findings not shown). of our study was the absence of high-affinity binding Although SP-containing neurons have been identi- sites in the rabbit iris and ciliary epithelia, known to fied in the retina of mammals,51 including humans,52 be the major site of the blood-aqueous barrier. It is very little is known about their functional role in this unlikely that the lack of binding sites in rabbit ciliary tissue. These neurons are present in most species in body may be due to occupied receptor sites since sec- amacrine cells whose somata are located at the border tions were washed in a preincubation medium for 15 of the INL and IPL (which send processes primarily to min before incubation with 125I-BHSP. Nevertheless, the IPL). Evidence for SP localization to ganglion we cannot exclude the possibility of the presence of cells has also been demonstrated in some species, par- lower-affinity receptors or other unknown subtypes in ticularly rabbits.53 The localization of SP binding sites ciliary process. Such sites may be absent or not detect- we found was similar to the pattern of SP-immunore- able by our autoradiographic procedures. However, active cells previously described for this region. Neo- these results suggest that, in the rabbit, the peptide is natal monosodium glutamate treatment of rats not directly involved in the blood-aqueous barrier (which induces degeneration of the inner layers of the disruption observed in ocular neurogenic inflamma- retina) has been reported to cause a marked reduction tion, as suggested by its weak and inconsistent effect. in 125I-BHSP retinal binding.19 Our autoradiographic In addition, we cannot exclude the possibility that SP findings also agreed with this experiment since most exerts some inflammatory actions in the eye through of the SP binding sites identified our study were con- an indirect mechanism; it has been demonstrated that centrated in the IPL and GCL. Several observations SP has a wide spectrum of inflammatory properties, suggest a neuromodulator role for SP in retinal func- in particular mast cell degranulation with release of tions; it has been found to elicit 3H-dopamine release histamine44 and arachidonic acid45 (and consequently from rabbit retinas18 and to exert excitatory effects on prostaglandin biosynthesis). Finally, the involvement carp cholinergic-sensitive ganglion cells activity.17 It of SP in ocular neurogenic inflammation cannot be has been also shown to stimulate the accumulation of generalized since it has been reported that the ocular inositol triphosphates in rabbit retinal cultures.54 The response to SP was species -dependent (cat, baboon, presence of SP binding sites in the inner retina is an or human iris are relatively insensitive to SP46). This additional indication that the peptide probably has species variation is well illustrated in the our study by some regulatory action in visual processing. the fact that the overall density of iridial SP binding sites was higher in the rabbit than in the rat. Key words: substance P, substance P receptor, autoradiography, eye The distribution of uveal SP binding sites and SP nerve terminals was not correlated well. In our study, regions that have relatively high densities of SP bind- Acknowledgments ing sites, such as the iris sphincter muscle, have been The authors thank Odile Flamand and Yves Issoulie for reported previously to have the highest amounts of SP technical assistance. immunoreactivity.47 In contrast, regions such as the ciliary processes or the iridial stroma, also known to contain SP immunoreactivity, did not have detect- References able SP binding sites. The existence of discrepancies 1. Duke-Elder PM and Duke-Elder WS: The vascular response of between the localization of receptors and the distribu- the eye. Proc R Soc Lond [Biol] 109:19, 1931. tion of fibers or neurotransmitters has been clearly 2. Cole DF and Unger WG: Prostaglandins as mediators for the demonstrated for SP in the brain.48 response of the eye to trauma. Exp Eye Res 17:357, 1973. The SP binding activity in the scleral vessels (Fig. 5) 3. Unger WG and Butler JM: Neuropeptides in the uveal tract. Eye 2:202, 1988. may be associated with the endothelium or vascular 4. Von Euler US and Gaddum JH: An unidentified depressor constituents. This may be attributed to the chemotac- substance in certain tissue extracts. J Physiol (Lond) 72:74, 49 tic peptide, f-Met-Leu-Phe (fMLP) or the phagocyto- 1931. sis promoting peptide, tuftsin,50 which share struc- 5. Lembeck F and Holzer P: Substance P as neurogenic mediator tural similarities with SP. Furthermore, SP has been of antidromic vasodilation and neurogenic plasma extravasa- tion. Naunyn Schmiedebergs Arch Pharmacol 310:175, 1979. shown to interact with the tuftsin receptors present on 6. Bill A, Stjernschantz J, Mandahl, Brodin E, and Nilsson G: the macrophage and polymorphonuclear leukocyte Substance P: Release on trigeminal nerve stimulation, effects 50 plasma membrane. However, under our experimen- in the eye. Acta Physiol Scand 106:371, 1979.

Downloaded from iovs.arvojournals.org on 10/01/2021 No. 6 OCULAR SUBSTANCE P BINDING SITES / Denis er ol 1901

7. Nishiyama A, Masuda K, and Mochizuki M: Ocular effects of cally active 125I-labeled substance P derivative to mouse mesen- substance P. Jpn J Ophthalmol 25:362, 1981. cephalic cells in primary culture. Mol Pharmacol 22:48, 1982. 8. Stone RA, Kuwayama Y, Laties AM, McGlinn AM, and 28. Mantyh PW, Johnson DJ, Boehmer CG, Catton MD, Vinters Schmidt ML. Guinea-pig ocular nerves contain a peptide of the HV, Maggio JE, Too HP, and Vigna SR: Substance P receptor cholecystokinin/gastrin family. Exp Eye Res 39:287, 1984. binding sites are expressed by glia in vivo after neuronal injury. 9. Stone RA and McGlinn AM: Calcitonin gene-related peptide Proc Natl Acad Sci U S A 86:5193, 1989. immunoreactive nerves in human and rhesus monkey eyes. 29. Kieselbach GF, Ragaut R, Knaus HG, Konig P, and Wieder- Invest Ophthalmol Vis Sci 29:305, 1988. mann CJ: Autoradiographic analysis of binding sites for 125I- 10. Unger WG, Terenghi G, Ghatei MA, Ennis KW, Butler JM, Bolton-Hunter-substance P in the human eye. Peptides 11:655, Zhang SQ, Too HP, Polak JM, and Bloom SR: Calcitonin 1990. gene-related peptide as a mediator of the neurogenic ocular 30. Maggio JE: Tachykinins. Annu Rev Neurosci 11:13, 1985. injury response. J Ocul Pharmacol 1:189, 1985. 31. Kanazawa I, Ogawa T, Kimura S, and Munekata E: Regional 11. Stone RA and Kuwayama Y: Substance P-like immunoreac- distribution of substance P, neurokinin alpha, and neurokinin tive nerves in the human eye. Arch Ophthalmol 103:1207, beta in the rat central nervous system. Neurosci Res 2:11,1984. 1985. 32. Saffroy M, Beaujouan JC, Torrens Y, Besseyre J, Bergstrom L, 12. Stjernschantz J, Sears M, and Stjernschantz L: Intraocular ef- and Glowinski J: Localization of tachykinin (NK1, NK2, NK3 fects of substance P in the rabbit. Invest Ophthalmol Vis Sci ligands) in the rat brain. Peptides 9:227, 1988. 20:53, 1981. 33. Butler JM, Powell D, and Unger WG: Substance P levels in 13. Ueda N, Muramatsu I, and Fujiwara M.: Capsaicin and brady- normal and sensorily denervated rabbit eyes. Exp Eye Res kinin induced substance P-ergic responses in the iris sphincter 30:311, 1980. muscle of the rabbit. J Pharmacol Exp Ther 230:469, 1984. 34. Tervo K, Tervo T, Eranko, L, Eranko O, and Cuello AC: Im- 14. Holmdahl G, Hakanson R, Leander S, Rosell S, Folkers K, and munoreactivity for substance P in the gasserian ganglion, oph- Sundler F: A substance P antagonist, (D-Pro2, D-Trp7-9) SP, thalmic nerve and anterior segment of the rabbit eye. Histo- inhibits inflammatory responses in the rabbit eye. Science chem J 13:435, 1981. 214:1029, 1981. 35. Beding-Barnekow B, Brodin E, and Hakanson R: Substance P, 15. Brecha N, Elred W, Kuljis RO, and Karten HJ: Identification neurokinin A and neurokinin B in the ocular response to injury and localization of biologically active peptides in the vertebrate in the rabbit. Br J Pharmacol 95:259, 1988. retina. In Progress in Retinal Research, Osborne N and Chader 36. Miller A, Costa M, Furness JB, and Chubb IW: Substance P J, editors. Pergamon Press, 1984, pp. 185-226. immunoreactive nerves supply the rat iris and cornea. Neuro- 16. Brecha NC, Stemini C, Anderson K, and Krause JE: Expres- sci Lett 23:243, 1981. sion and cellular localization of substance P/neurokinin A and 37. Shimizu Y, Kuwayama Y, Fukuda M, Ishimoto I, Shiosaka S, neurokinin B mRNAs in the rat retina. Vision Neuroscience Inagaki S, Takagi H, Sakanaka M, Senba E, Kawai Y, Takat- 3:527, 1989. suki K, and Tohyama M: Localization of substance P-like im- 17. Glickman RD and Adolph AR: Acetylcholine and substance P: munoreactivity in the anterior segment of squirrels: An immu- Action via distinct receptors on carp retinal ganglion cells. In- nohistochemical analysis. Invest Ophthalmol Vis Sci 22:259, vest Ophthalmol Vis Sci 22:804, 1982. 1982. 18. Tsang D: Effect of substance P on dopamine release in rat ret- 38. Soloway MR, Stjernschantz J, and Sears M: The miotic effect ina. In Contemporary Themes in Biochemistry, Kon OL, of substance P on the isolated iris. Invest Ophthalmol Vis Sci Chung MCM, Hwang PLH, Leong SF, Loke KH, Thiayagara- 21:47, 1981. jah P, and Wong PTH, editors. 1986, pp. 588-589. 39. Oksala O: Effects of calcitonin gene-related peptide and sub- 19. Lee CM and Cheung WT: Effects of neonatal monosodium stance P on regional blood flow in the cat eye. Exp Eye Res glutamate treatment on substance P binding sites in the rat 47:283, 1988. retina. Neurosci Lett 92:310, 1988. 40. Krootila K: CGRP in relation to neurogenic inflammation and 20. Osborne NN: Substance P in the bovine retina: Localization, cAMP in the rabbit eye. Exp Eye Res 47:307, 1988. identification, release, uptake and receptor analysis. J Physiol 41. Kuwayama Y and Stone RA: Distinct substance P and calci- (Lond) 349:83, 1984. tonin gene-related peptide immunoreactive nerves in the 21. Hirata H, Baba S, Mishima H, and Choschi K: Specific binding guinea pig eye. Invest Ophthalmol Vis Sci 28, 1947, 1987. 125 of I-substance P in the bovine iris-ciliary body. Nippon 42. Wahlestedt C, Beding B, Ekman R, Oksala O, Stjernschantz J, Ganka Gakkai Zasshi 10:935, 1983. and Hakanson R: Calcitonin gene-related peptide in the eye: 22. Too HP, Unger WG, and Hanley MR: Evidence for multiple Release by sensory nerve stimulation and effects associated tachykinin receptor subtypes on the rabbit iris sphincter mus- with neurogenic inflammation. Regul Pept 16:107, 1986. cle. Mol Pharmacol 33:64, 1988. 43. Suzuki R and Kobayashi S: Different effects of substance P and 23. Mantyh PW, Gates T, Mantyh CR, and Maggio JE: Autoradio- vasoactive intestinal peptide on the motor function of bovine graphic localization of tachykinin receptor binding sites in the intraocular muscles. Invest Ophthalmol Vis Sci 24:1566, 1983. rat brain and peripheral tissues. J Neurosci 1:258, 1989. 24. Pernow B: Substance P. Pharmacol Rev 35:85, 1983. 44. Hagermark O, Hokfelt T, and Pernow B: Flare and itch in- 25. Regoli D, Drapeau G, Dion S, and D'Orleans-Juste P: Recep- duced by substance P in human skin. J Invest Dermatol tors for substance P and related neurokinins. Pharmacology 71:233, 1978. 38:1, 1989. 45. Yousufzai SYK, Akhtar RA, and Abdel-Latif AA: Effects of 26. Stephenson JA, Summers RJ, and Burcher E: Autoradio- substance P on inositol triphosphate accumulation, on con- graphic localization and characterization of substance P bind- tractile responses and on arachidonic acid release and prosta- ing in dog kidney. Eur J Pharmacol 142:391, 1987. glandin biosynthesis in rabbit iris sphincter muscle. Exp Eye 27. Beaujouan JC, Torrens Y, Daguet MC, Glowinski J, and Pro- Res 43:215, 1986. chiantz A: Specific binding of an immunoreactive and biologi- 46. Unger WG and Tighe J: The response of the isolated iris

Downloaded from iovs.arvojournals.org on 10/01/2021 1902 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Moy 1991 Vol. 32

sphincter muscle of various mammalian species to substance P. tetrapeptide sequence. Biochem Biophys Res Commun Exp Eye Res 39:677, 1984. 94:1445, 1980. 47. Cole DF, Bloom SR, Burnstock, Butler JM, McGregor GP, 51. Brecha N: Retinal neurotransmitters. In Chemical Neuroanat- Saffrey MJ, Unger WG, and Zhang SQ: Increase in SP-immun- omy, Emson PC, editor. New York, Raven Press, 1983, pp. oreactivity in nerve fibres of rabbit iris and ciliary body one to 85-129. four months following sympathetic denervation. Exp Eye Res 37:191, 1983. 52. Tornqvist K and Ehinger B: Peptide immunoreactive neurons 48. Herkenham M: Mismatches between neurotransmitter and re- in the human retina. Invest Ophthalmol Vis Sci 29:680, 1988. ceptor localizations in brain: Observations and implications. 53. Brecha NC, Johnson D, Bolz J, Sharma S, Parnavelas JG, and Neuroscience 23:1, 1987. Lieberman AR: Substance P immunoreactive retinal ganglion 49. Marasco WA, Showell HJ, and Becker EL: Substance P binds cells and their central axon terminals in the rabbit. Nature to the formylpeptide chemotaxis receptor on the rabbit neutro- 327:155, 1987. phil. Biochem Biophys Res Commun 99:1065, 1981. 54. Osborne NN and Ghazi H: The effect of substance P and other 50. Bar-Shavit Z, Goldman R, Stabinsky Y, Gottlieb P, Fridkin M, tachykinins on inositol phospholipid hydrolysis in rabbit ret- Teichberg VI, and Blumberg S: Enhancement of phagocytosis: ina, superior colliculus and retinal cultures. Vision Res 29:754, A newly found activity of substance P residing in its N-terminal 1989.

Downloaded from iovs.arvojournals.org on 10/01/2021