localization and characterization of substance p binding...

Investigative Ophthalmology & Visual Science, Vol. 32, No. 6, May 1991Copyright © Association for Research in Vision and Ophthalmology

Localization and Characterization of Substance PBinding 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 andrats using an in vitro autoradiographic method with l2SI-Bolton Hunter SP (BHSP). Autoradiogramswere generated by apposing 10-20/im-thick cryostat eye sections to 3H-Hyperfilm or liquid emulsionand quantified by means of image-analysis procedures. Kinetic studies showed that equilibrium wasreached after a 75-min incubation at room temperature. In rat retina, specific binding corresponding toapproximately 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 twoneurokinins (NK) A and B competed in a concentration-dependent manner for retinal sites labeled by125I-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 sphinctermuscle, choroid, and retina. In rats, detectable amounts of SP-binding sites were also expressed in thecorneal epithelium and iridial stroma. Quantitative analysis of the autoradiograms revealed that thehighest densities of 125I-BHSP binding sites were localized in the iris sphincter muscle in rabbits andthe inner retina in rats. Invest Ophthalmol Vis Sci 32:1894-1902,1991

Numerous neurogenic mediators released in the an-terior segment of the eye by ocular injury, trauma, ornoxious stimulation are known to elicit inflammatoryeffects, such as conjunctival hyperemia, miosis, rise inintraocular pressure, and disruption of the blood-aqueous barrier.' If some of these biologic effects aredemonstrated to be mediated directly by metabolitesfrom the arachidonic acid cascade (released from theiris and the ciliary body),2 it is now widely recognizedthat neural pathways also participate in the initiationof such inflammatory events by releasing neuropep-tides from sensory afferent nerves in the uveal tract.3

Substance P (SP), an undecapeptide isolated from in-testine in 1931,4 was first proposed as a neurogenicmediator of antidromic vasodilation and plasma ex-travasation at the peripheral level5 and as a majorcomponent in the neurogenic ocular injury re-sponses.6'7 Later, other biologically active substances,

From the *1NSERM U55 and tDepartment of Ophthalmology,Hopital Saint Antoine, Paris, JRhone-Poulenc Sante, Departmentof Biology, Vitry-Sur-Seine, and the §Department of Pharmacol-ogy, Faculte de Medecine, Nice, France.

Philippe Denis was a recipient of INSERM (Poste d'accueil).Submitted for publication: September 25, 1990; accepted .Reprint requests: Philippe Denis, INSERM U55, 184, rue du

Faubourg Saint Antoine, 75012, Paris, France.

such as calcitonin gene-related peptide (CGRP) orcholecystokinin, have also been identified in ocularsensory structures8'9 and shown to play a functionalrole in the neurogenic inflammation (particularlyblood-aqueous barrier breakdown for CGRP.l0) How-ever SP involvement in neurogenic inflammation issuggested by several pieces of evidence. First, nerveendings with immunoreactivity to SP are found in theuvea of several species, including humans," mainly inclose association with the sphincter muscle of the irisand the smooth blood vasculature in the ciliary body.Second intracameral administration of SP induces adose-dependent, nonmuscarinic pupil constrictionassociated with aqueous flare and an increase in intra-ocular pressure in the rabbit eye.6 Third electrical stim-ulation of the trigeminal ganglion or intracameral ad-ministration of capsaicin (both responsible for SP re-lease in the anterior segment) are able to mimicSP-induced miosis.61213 Fourth, (D-Pro2, D-Trp7-9)-SP, a SP antagonist, counteracts this phenomenon inrabbits and could therefore inhibit the ocular inflam-matory response to laser iridial burns.14

Biochemical and immunohistochemical studieshave localized SP in various vertebrate retinas.15 Cel-lular expression of SP-encoding mRNA was found re-cently in the rat retina using RNA blot and in situhybridization.16 Although physiologic studies indi-

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No. 6 OCULAR SUBSTANCE P BINDING SITES / Denis er al 1895

cate that SP has a neuromodulator action on ganglioncells in fish17 and in dopamine release from the retinain the rat,18 the role of SP immunoreactive neurons inthe processing of visual information is not yet fullyunderstood.

The presence of ocular SP binding sites was sug-gested previously by conventional binding techniquesusing membranes obtained from rat and bovine ret-ina1920 or bovine and rabbit iris,2122 but the precisedistribution of these receptor sites has not yet beeninvestigated extensively. One autoradiographic reportbriefly mentioned the presence of binding sites in therat retina,23 but no quantitative data or informationon the pharmacologic profile of SP binding were pre-sented. It is important to localize these binding sitessince it is believed that most SP biologic actions arereceptor -mediated.24 We therefore characterized anddetermined the anatomic localization of SP bindingsites in rat and rabbit eyes using quantitative in vitroautoradiographic methods. We have used Bolton-Hunter SP (125I-BHSP; Amersham, les Ulis, France),a radiolabeled analogue of the tachykinin which hasbeen extensively used in other organs to label SP re-ceptors.25"27

Materials and Methods

Tissue Preparation

New Zealand albino rabbits (weighing 3-3.5 kg)were killed by injection of a lethal dose of sodiumpentobarbital and Wistar rats (weighing 200-250 g),by decapitation. The eyes were removed, immersed inTissue Tek medium (Miles Scientific, Naperville, IL),frozen in isopentane cooled (-40°C) in liquid nitro-gen, and stored at -80°C. Just before sectioning, thetissues were warmed to -20°C, and sections (20-fxmthick) were cut with a cryostat (Bright), thaw-mounted onto gelatin-coated glass slides, and storedat -80°C until use. Before incubations, sections wereallowed to thaw at room temperature. All investiga-tions described in this paper were done in accordancewith the ARVO Resolution on the Use of Animals inResearch.

Binding Conditions

Preliminary experiments showed that preincuba-tion was found to increase specific binding. Thus,slides were washed in a preincubation medium (Tris-HC1 50 mM, pH 7.4, containing 0.2 g/1 of bovineserum albumin) at room temperature for 15 min be-fore incubation with radioligand. After the washingstep, the slides were then incubated at room tempera-ture in a solution of 65 pM 125I-BHSP (2000 Ci/mmol) [Bolton-Hunter is 3-(p-hydroxy-m-(125I)iodo-phenyl)-propionyl) in 50 mM Tris -HC1, pH 7.4, con-

taining 10 mM MgCl2,2 g/1 of bovine serum albumin,40 mg/1 of bacitracin, 5 mg/1 of leupeptin, and 4 mg/1of bestatin. Nonspecific binding was determined onalternate sections in the presence of 10 ju.M unlabeledpeptide (SP) added in the incubation medium. Thespecificity of the binding was studied by incubatingsections with increasing concentrations of peptidesrelated to SP, such as senktide, septide, spantide,SP methylester, neurokinin A (NKA), neurokininB (NKB), and synthetic fragments of SP (SP,_4 andSP4_n). Bacitracin, leupeptin, and bestatin were ob-tained from Sigma (St. Louis, MO) and SP-relatedpeptides from Bachem (Bubendorf, Switzerland).After incubation with the radioligand, the sectionswere rinsed four times for 1 min each in the preincu-bation buffer at 4°C, dipped for 20 sec into distilledwater, and quickly dried using a stream of cold air forautoradiography.

Autoradiographic Experiments

The sections were preincubated as described andincubated with 65 pM 125I-BHSP for 90 min at roomtemperature. After washing, the dry slides were storedin a Kodak X-ray cassette (Rochester, NY) in tightapposition to a tritium-sensitive film (3H-Hyperfilm;Amersham) and allowed to expose for 1 week in dark-ness. After exposure, the films were developed in Ko-dak D19 for 3 min and fixed. To identify the localiza-tion of the binding sites, the eye sections were coun-terstained with hematoxylin and eosin and coverslipped with Fluka (Chemika, Buchs, Switzerland).

To investigate precisely the cellular localization ofSP binding sites, an autoradiographic technique wasused at light microscopic resolution. Briefly, after in-cubation and washings, selected slides were treatedwith a 30-min bath of 4% glutaraldehyde at 4°C to fixcovalently the radioligand to its binding site, defattedin several baths of increasing concentrations of alco-hol (75-100%) and xylene, and then dipped into liq-uid nuclear emulsion (LM1,; Amersham). Prelimi-nary experiments showed that this treatment did notsignificantly alter 125I-BHSP binding. After an expo-sure period of 10 days in darkness, the emulsiondipped microautoradiograms were developed andfixed as described. Corresponding sections were coun-terstained and examined under a light microscope.Light- and dark-field photomicrographs were takenfrom the stained sections and silver grains, respec-tively.

Data Analysis

Film macroautoradiograms were analyzed by com-puter-based densitometry. The optical density of theautoradiogram was quantified by means of an image


1896 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1991 Vol. 32

analyzer (BIOCOM RAG 200, les Ulis, France).Briefly, autoradiograms were digitized, and each graindensity was assigned a relative optical density value.For maximal binding capacity determination, theserelative optical density values were then converted tocorresponding commercial 125I Amersham standards,and the results were expressed in fmol/mg tissueequivalent. Amersham's microscales are supplied asslices of several layers of polymer containing a rangeof increasing 1251 concentrations. Since tissue sec-tions are coexposed with iodinated plastic standards,we can do quantitative densitometry using a com-puter-assisted system. Tissue-equivalent values wereprovided based on calibration using intact brain graymatter. In all cases, specific binding was defined bysubtracting nonspecific binding (obtained in the pres-ence of 10 iiM of unlabeled SP) from total binding.Classic computer analysis was used for the biochemi-cal determination of the binding parameters (dissocia-tion constant [Kd] and concentrations which inhibit50% of the specific binding [IC50]) on rat retina sec-tions. The kinetics of I25I-BHSP binding was assumedto follow a bimolecular association model (second-order kinetics).

Results

Characterization of 125I-BHSP BindingKinetic analysis done by densitometry on rat reti-

nal sections indicated that the association of 125I-

8O -

5O TOO 15OTime(min)

Fig. 1. Association and dissociation of I25I-BHSP binding to ratretina sections. Retina sections were incubated with 65 pM 125I-BHSP at room temperature for various times. The curve representsspecific binding that was calculated as the difference between bind-ing in the presence and absence of 10-̂ M unlabeled substance P(SP). Dissociation was initiated by the addition of 10-juM unlabeledSP at 120 min, a time at which the association of the ligand hadreached the equilibrium. Values are given as the mean of triplicatedeterminations in a typical experiment.

o00

00

11 10 9 8SP concentration

7 6Log.M

Fig. 2. Competition between 125I-BHSP and unlabeled SP forbinding to rat retina sections. 65 pM 125I-BHSP were incubatedalone or in the presence of increasing concentrations of unlabeledpeptide SP at room temperature for 90 min. 125I-BHSP binding isexpressed as a percent of total binding. Values are given as the meanof triplicate determinations.

BHSP resulted in a time-dependent increase in bind-ing (Fig. 1). At room temperature, specific bindingreached a plateau in approximately 75 min; nonspe-cific binding was not significantly increased. About90% of total binding was specific at 75 min. A routineincubation time of 90 min was then adopted for allsubsequent experiments. At equilibrium, bound 125I-BHSP could be dissociated specifically from its bind-ing sites by incubating sections with an excess (10 ixM)of unlabeled peptide (Fig. 1). The half-time for dissoci-ation of 125I-BHSP was about 15 min.

We investigated competition studies between io-dinated SP and increasing concentrations of unla-beled peptide (Fig. 2). These results indicated that SPinhibited 125I-BHSP competitively and with very highaffinity. In the range of concentrations studied (10"u

to 10~6 M), IC50 values were 0.17 nM. Curve analysisobtained (obtained by regression lines computerizedby means of the least-squares method) indicated that125I-BHSP bound to high-affinity binding sites inrat retina with an apparent Kd estimated at 0.13± 0.02 nM.

Competition studies with the tachykinins NKA,NKB, and analogues or fragments of SP were done inattempt to establish the type of site identified by I25I-BHSP in the rat retina. The order of potency for inhi-bition of binding was SP > NKA > NKB (Fig. 3, Ta-ble 1). A similar ranking was obtained when rabbiteye sections were incubated with the tachykinins atconcentrations in the range of 10"8 to 10~5 M (datanot shown). Displacement curves showed that specific


No. 6 OCULAR 5UDSTANCE P BINDING SITES / Denis er al 1897

. S PA S P 4-11• NKA0 S Pl-4ANKB

11 10 9 8 7 6Peptide concentration -Log M

Fig. 3. Pharmacology of 125I-BHSP binding. 65 pM 125I-BHSPwere incubated alone or in the presence of increasing concentra-tions of unlabeled peptides at room temperature for 90 min. I25I-BHSP binding is expressed as a percent of initial binding. Values aregiven as the mean of four determinations.

binding of I25I-BHSP was strongly inhibited bySP,_n (IC50=, 0.17 ± 0.02) (Fig. 2) and to a lesserextent by SP4_n and NKA (IC50=, 3.39 and 8.04 nM,respectively). On the other hand, senktide and septidewas ineffective, and SP^, NKB, and spantide hadsome potency to inhibit retinal I25I-BHSP binding(Table 1).

Autoradiographic Experiments

Representative autoradiograms of 125I-BHSP bind-ing sites are shown in Figure 4-6. In the rat anteriorsegment, SP binding sites were found in the iris,mainly in the sphincter region (Fig. 4, Panel a). Analy-sis of histologic sections confirmed that labeling onthe pupil margin coincided with the iris muscular tis-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.In contrast, moderate 125I-BHSP binding densitieswere noted over the other iridial structures and in thecorneal epithelium. No labeling occurred in struc-tures such as the sclera and the ciliary processes. Bind-ing seen on the lens fibers was not considered to bespecific since SP did not displace 125I-BHSP labeling.In the posterior segment, the retina showed intensivelabeling; low concentrations of binding were noted inthe choroid. Binding sites were not uniformly distrib-uted throughout the retina since a slight decrease ofbinding at the ora serrata was observed. Autoradio-grams of the emulsion-dipped slides provided detailedhistologic resolution of the retinal SP binding sites(Fig. 5). Silver grains were localized primarily in theinner plexiform (IPL) and ganglion cell layers (GCL).A few scattered grains were also detected in the outer

plexiform layer and proximal inner nuclear layer(INL). Much lower SP binding was present in otherregions, particularly in the outer retina (outer nuclearlayer and photoreceptor cell layer). Similarly, verylow, but significant, concentrations of SP bindingsites were seen in the choroidal tissue. Some scleralblood vessels also exhibited some binding activity(Fig. 5, see arrows).

In the rabbit eye (Fig. 6), the topographic distribu-tion of SP binding sites was similar to that found inthe rat although there were some quantitative differ-ences (Table 2). Very high labeling was seen in associa-tion with the iridial sphincter muscle; no labeling wasfound either in the cornea, iris stroma, or ciliary body.Low binding was also identified in the choroid. As inthe rat, lens fibers gave important nonspecific bind-ing, ie, not displaceable in the presence of an excess ofunlabeled peptide. The retina also had high densitiesof specific SP binding sites, mainly concentrated inthe IPL and GCL, as described in the rat.

Discussion

These results demonstrate that several structuresfrom albino rat and rabbit eyes (mainly the retina andthe iris sphincter muscle) have the capacity to bindspecifically a derivative analogue of SP, I25I-BHSP.The characteristics of I25I-BHSP binding in the ratretina strongly suggest that the ligand binds to SP re-ceptors.

The apparent Kd of the high-affinity (0.13 nM)binding site identified in this study was similar tothose reported in rat retina homogenates (0.2 nM),19

in rabbit optic sections (0.54 nM),28 and in humanretina (0.27 nM).29 Recent studies have described theexistence of multiple tachykinin receptors in the ratretina.23 The agent, SP, belongs to a group of closely

Table 1.

Peptides

Substance PSubstance P^,,Neurokinin ASubstance P,_4Neurokinin BSpantideSubstance P methyl esterSeptideSenktide

ICso (nM)

0.17 ±0.023.39 ± 0.408.04 ± 0.57

35 ±4.3452.8 ± 4.06

53 ± 1.5354 ± 6.34>1000>1000

Displacement of 125I-BHSP binding on rat retina sections by substance P(SP), peptides related to SP, and synthetic fragments of SP. Sections wereincubated for 90 min at room temperature with 65 pM I251-BHSP and in-creasing concentrations (10"" to 10~6 M) of unlabeled peptides. Nonspecificbinding of the radioligand was determined in the presence of 10-MM unla-beled SP. IC50 is defined as the molar concentration of the tested drug thatdisplaced 50% of the specific binding of I25I-BHSP on retinae sections. Autora-diograms were analyzed by computer-based densitometry with an image ana-lyser. Values are given as the mean of triplicate determinations.



ISM

/|SM

the mammalian tachykinins have been described,namely SP (or NK-1) receptors, found in both centraland peripheral tissues, NKA (or NK-2) receptors inperipheral tissues, and NKB (or NK-3) receptorsmainly detectable in the central nervous system.23'32

Our data suggest that 125I-BHSP binds preferentiallyto NK-1 receptor sites; SP is the most potent tachy-kinin in inhibiting 25I-BHSP binding and the order ofpotency of the competitors for 125I-BHSP binding wasSP > NKA > NKB. The IC50 values calculated forNKA and NKB were somewhat lower than thosefound for the displacement of I25I-BHSP binding in apreliminary report.19 Variations in the methods, in-cluding the incubation buffer and the lower ligandconcentration used in our study (65 pM), may be thecause of these differences. It is unlikely that I25I-BHSPbinds to NKB receptors since senktide, a highly selec-tive 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 65pM I251-BHSP alone (A) or in presence of 10-^M unlabeled SP todetermine nonspecific binding (B). Autoradiograms were generatedby 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.

related peptides known as tachykinins, all of whichcontain a common C-terminal amino acid sequence:Phe-X-Gly-Leu-Met-NH2.

30 In 1983, two additionaltachykinins, NKA and NKB, were isolated in mam-malian tissues.31 To date, three distinct receptors for

BFig. 5. Microautoradiograms of I25I-BHSP binding in albino rat

posterior segment. Dark field (A) and bright field (B) were obtainedafter incubation of 65 pM '"l-BHSP alone. Silver grains visualizedin the retina and choroid indicated specific SP binding sites sincelabeling disappeared after incubation with an excess of unlabeledpeptide (not shown). GCL, ganglion cell layer; IPL, inner plexiformlayer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL,outer nuclear layer; SV, scleral vessel. Magnification X500.


No. 6 OCULAR SUBSTANCE P BINDING SITES / Denis er ol 1899

ISM

B

Fig. 6. Autoradiograms of I25I-BHSP binding in albino rabbitanterior segment. Sections were incubated for 90 min at room tem-perature with 65 pM I25I-BHSP alone (A) or in presence of 10-^Munlabeled SP (B). Auloradiograms were generated by apposition to3H-Hyperfilm for 7 days. (C) Histological section, C, cornea; ISM,iris sphincter muscle; CP, ciliary processes; L, lens. MagnificationX3,

site. Furthermore, as described for SP binding sites inother tissues, l9'25>26 affinity is encoded in the carboxyterminal of SP since SP,_, j and SP4_n had the greatestpotency in inhibiting I25I-BHSP binding.

Autoradiographic visualization of 125I-BHSP bind-ing sites in the anterior segment of the eye shows aselective pattern of distribution. In both species, SP

binding sites were found to be mainly associated withthe iris sphincter muscle although other iridial struc-tures were slightly labeled in the rat eye. The presenceof SP was reported in sensory neurons of the anterioruvea originating from the trigeminal ganglion,3334

and a number of investigators extensively studied theinvolvement of the peptide in the acute irritation re-sponse of the eye. Besides its vasodilator properties,early experiments reported that the tachykinin pep-tide induces an increase in intraocular pressure andmoderate alterations in the blood-aqueous barrier inrabbit eyes.6'714'35 Subsequent studies observed thatthese effects were relatively weak and inconsistentand that the rise in intraocular pressure was mainlydue to a miosis-induced pupillary blockade since itwas abolished in part by peripheral iridectomies.12

Despite its wide distribution in the uveal tract inmany species,11'33'34'36'37 SP has been proposed to beonly responsible for the miotic component of the anti-dromic ocular injury response.12'3538 The other partsof the inflammatory response (elevated intraocularpressure and blood-aqueous barrier breakdown) arethought to be mediated by other mechanisms; in ani-mals pretreated with prostaglandin inhibitors, injec-tion of SP caused only miosis.12 The release of CGRPfrom sensory nerves has also been shown to inducemarked inflammatory effects in cat39 and rabbit40

eyes. The two neuropeptides are partially colocalizedin terminal endings arising from the trigeminal cells41

and are released together into aqueous humor duringthe antidromic response. In addition, SP-inducedmiosis is potentiated by CGRP,42 suggesting biologicinteractions between the two peptides. Our autoradio-graphic results provide essential information con-cerning the role of SP in the ocular anterior segment;they demonstrate the presence of high-affinity bind-ing sites for SP associated with the iridial sphinctermuscle. They may also suggest that the miotic re-sponse to SP in rabbits may be due to a direct action

Table 2.

Area

CorneaIris stromaIris sphincter muscle

Retina

ratratrabbitratrabbitrat

Specific l2iI-BHSP bound(fmol/mg tissue equivalent)

0.12 ±0.010.11 ±0.012.93 ± 0.420.17 ±0.010.95 ±0.170.63 ± 0.07

Regional distribution of specific BHSP binding sites in the albino rat andrabbit eye. Results were obtained by transformation of optical densities asdescribed in the text to fmol/mg tissue equivalent ± SEM and were obtainedfrom at least 10 determinations throughout the area under examination.Nonspecific binding was subtracted from each total value, so that data areexpressed as specific binding.



on the iris muscle through specific receptors. No bind-ing sites was found in dilator or ciliary muscles, cor-roborating the fact that SP has no contractile effect onthese tissues.3843 One of the most interesting findingsof our study was the absence of high-affinity bindingsites in the rabbit iris and ciliary epithelia, known tobe the major site of the blood-aqueous barrier. It isunlikely that the lack of binding sites in rabbit ciliarybody may be due to occupied receptor sites since sec-tions were washed in a preincubation medium for 15min before incubation with 125I-BHSP. Nevertheless,we cannot exclude the possibility of the presence oflower-affinity receptors or other unknown subtypes inciliary process. Such sites may be absent or not detect-able by our autoradiographic procedures. However,these results suggest that, in the rabbit, the peptide isnot directly involved in the blood-aqueous barrierdisruption observed in ocular neurogenic inflamma-tion, as suggested by its weak and inconsistent effect.In addition, we cannot exclude the possibility that SPexerts some inflammatory actions in the eye throughan indirect mechanism; it has been demonstrated thatSP has a wide spectrum of inflammatory properties,in particular mast cell degranulation with release ofhistamine44 and arachidonic acid45 (and consequentlyprostaglandin biosynthesis). Finally, the involvementof SP in ocular neurogenic inflammation cannot begeneralized since it has been reported that the ocularresponse to SP was species -dependent (cat, baboon,or human iris are relatively insensitive to SP46). Thisspecies variation is well illustrated in the our study bythe fact that the overall density of iridial SP bindingsites was higher in the rabbit than in the rat.

The distribution of uveal SP binding sites and SPnerve terminals was not correlated well. In our study,regions that have relatively high densities of SP bind-ing sites, such as the iris sphincter muscle, have beenreported previously to have the highest amounts of SPimmunoreactivity.47 In contrast, regions such as theciliary processes or the iridial stroma, also known tocontain SP immunoreactivity, did not have detect-able SP binding sites. The existence of discrepanciesbetween the localization of receptors and the distribu-tion of fibers or neurotransmitters has been clearlydemonstrated for SP in the brain.48

The SP binding activity in the scleral vessels (Fig. 5)may be associated with the endothelium or vascularconstituents. This may be attributed to the chemotac-tic peptide, f-Met-Leu-Phe (fMLP)49 or the phagocyto-sis promoting peptide, tuftsin,50 which share struc-tural similarities with SP. Furthermore, SP has beenshown to interact with the tuftsin receptors present onthe macrophage and polymorphonuclear leukocyteplasma membrane.50 However, under our experimen-

tal conditions, competition studies with increasingconcentrations of tuftsin (10~4 to 10~7 M) showed that125I-BHSP binding was not inhibited by tuftsin (datanot shown).

Although SP-containing neurons have been identi-fied in the retina of mammals,51 including humans,52

very little is known about their functional role in thistissue. These neurons are present in most species inamacrine cells whose somata are located at the borderof the INL and IPL (which send processes primarily tothe IPL). Evidence for SP localization to ganglioncells has also been demonstrated in some species, par-ticularly rabbits.53 The localization of SP binding siteswe found was similar to the pattern of SP-immunore-active cells previously described for this region. Neo-natal monosodium glutamate treatment of rats(which induces degeneration of the inner layers of theretina) has been reported to cause a marked reductionin 125I-BHSP retinal binding.19 Our autoradiographicfindings also agreed with this experiment since mostof the SP binding sites identified our study were con-centrated in the IPL and GCL. Several observationssuggest a neuromodulator role for SP in retinal func-tions; it has been found to elicit 3H-dopamine releasefrom rabbit retinas18 and to exert excitatory effects oncarp cholinergic-sensitive ganglion cells activity.17 Ithas been also shown to stimulate the accumulation ofinositol triphosphates in rabbit retinal cultures.54 Thepresence of SP binding sites in the inner retina is anadditional indication that the peptide probably hassome regulatory action in visual processing.

Key words: substance P, substance P receptor, autoradiogra-phy, eye

Acknowledgments

The authors thank Odile Flamand and Yves Issoulie fortechnical assistance.

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