Download Localization and characterization of substance P binding

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
125
I-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
such as calcitonin gene-related peptide (CGRP) or
cholecystokinin, have also been identified in ocular
sensory structures8'9 and shown to play a functional
role in the neurogenic inflammation (particularly
blood-aqueous barrier breakdown for CGRP.l0) However SP involvement in neurogenic inflammation is
suggested by several pieces of evidence. First, nerve
endings with immunoreactivity to SP are found in the
uvea of several species, including humans," mainly in
close association with the sphincter muscle of the iris
and the smooth blood vasculature in the ciliary body.
Second intracameral administration of SP induces a
dose-dependent, nonmuscarinic pupil constriction
associated with aqueousflareand an increase in intraocular pressure in the rabbit eye.6 Third electrical stimulation of the trigeminal ganglion or intracameral administration of capsaicin (both responsible for SP release 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 inflammatory response to laser iridial burns.14
Biochemical and immunohistochemical studies
have localized SP in various vertebrate retinas.15 Cellular expression of SP-encoding mRNA was found recently in the rat retina using RNA blot and in situ
hybridization.16 Although physiologic studies indi-
Numerous neurogenic mediators released in the anterior segment of the eye by ocular injury, trauma, or
noxious stimulation are known to elicit inflammatory
effects, such as conjunctival hyperemia, miosis, rise in
intraocular pressure, and disruption of the bloodaqueous barrier.' If some of these biologic effects are
demonstrated to be mediated directly by metabolites
from the arachidonic acid cascade (released from the
iris and the ciliary body),2 it is now widely recognized
that neural pathways also participate in the initiation
of such inflammatory events by releasing neuropeptides from sensory afferent nerves in the uveal tract.3
Substance P (SP), an undecapeptide isolated from intestine in 1931,4 was first proposed as a neurogenic
mediator of antidromic vasodilation and plasma extravasation at the peripheral level5 and as a major
component in the neurogenic ocular injury responses.6'7 Later, other biologically active substances,
From the *1NSERM U55 and tDepartment of Ophthalmology,
Hopital Saint Antoine, Paris, JRhone-Poulenc Sante, Department
of Biology, Vitry-Sur-Seine, and the §Department of Pharmacology, 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.
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OCULAR SUBSTANCE P BINDING SITES / Denis er al
No. 6
cate that SP has a neuromodulator action on ganglion
cells in fish17 and in dopamine release from the retina
in the rat,18 the role of SP immunoreactive neurons in
the processing of visual information is not yet fully
understood.
The presence of ocular SP binding sites was suggested previously by conventional binding techniques
using membranes obtained from rat and bovine retina1920 or bovine and rabbit iris,2122 but the precise
distribution of these receptor sites has not yet been
investigated extensively. One autoradiographic report
briefly mentioned the presence of binding sites in the
rat retina,23 but no quantitative data or information
on the pharmacologic profile of SP binding were presented. It is important to localize these binding sites
since it is believed that most SP biologic actions are
receptor -mediated.24 We therefore characterized and
determined the anatomic localization of SP binding
sites in rat and rabbit eyes using quantitative in vitro
autoradiographic methods. We have used BoltonHunter SP (125I-BHSP; Amersham, les Ulis, France),
a radiolabeled analogue of the tachykinin which has
been extensively used in other organs to label SP receptors.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 sodium
pentobarbital and Wistar rats (weighing 200-250 g),
by decapitation. The eyes were removed, immersed in
Tissue Tek medium (Miles Scientific, Naperville, IL),
frozen in isopentane cooled (-40°C) in liquid nitrogen, and stored at -80°C. Just before sectioning, the
tissues were warmed to -20°C, and sections (20-fxm
thick) were cut with a cryostat (Bright), thawmounted onto gelatin-coated glass slides, and stored
at -80°C until use. Before incubations, sections were
allowed to thaw at room temperature. All investigations described in this paper were done in accordance
with the ARVO Resolution on the Use of Animals in
Research.
Binding Conditions
Preliminary experiments showed that preincubation 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 bovine
serum albumin) at room temperature for 15 min before incubation with radioligand. After the washing
step, the slides were then incubated at room temperature in a solution of 65 pM 125I-BHSP (2000 Ci/
mmol) [Bolton-Hunter is 3-(p-hydroxy-m-(125I)iodophenyl)-propionyl) in 50 mM Tris -HC1, pH 7.4, con-
1895
taining 10 mM MgCl2,2 g/1 of bovine serum albumin,
40 mg/1 of bacitracin, 5 mg/1 of leupeptin, and 4 mg/1
of bestatin. Nonspecific binding was determined on
alternate sections in the presence of 10 ju.M unlabeled
peptide (SP) added in the incubation medium. The
specificity of the binding was studied by incubating
sections with increasing concentrations of peptides
related to SP, such as senktide, septide, spantide,
SP methylester, neurokinin A (NKA), neurokinin
B (NKB), and synthetic fragments of SP (SP,_4 and
SP4_n). Bacitracin, leupeptin, and bestatin were obtained from Sigma (St. Louis, MO) and SP-related
peptides from Bachem (Bubendorf, Switzerland).
After incubation with the radioligand, the sections
were rinsed four times for 1 min each in the preincubation buffer at 4°C, dipped for 20 sec into distilled
water, and quickly dried using a stream of cold air for
autoradiography.
Autoradiographic Experiments
The sections were preincubated as described and
incubated with 65 pM 125I-BHSP for 90 min at room
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;
Amersham) and allowed to expose for 1 week in darkness. After exposure, the films were developed in Kodak D19 for 3 min and fixed. To identify the localization of the binding sites, the eye sections were counterstained with hematoxylin and eosin and cover
slipped with Fluka (Chemika, Buchs, Switzerland).
To investigate precisely the cellular localization of
SP binding sites, an autoradiographic technique was
used at light microscopic resolution. Briefly, after incubation and washings, selected slides were treated
with a 30-min bath of 4% glutaraldehyde at 4°C to fix
covalently the radioligand to its binding site, defatted
in several baths of increasing concentrations of alcohol (75-100%) and xylene, and then dipped into liquid nuclear emulsion (LM1,; Amersham). Preliminary experiments showed that this treatment did not
significantly alter 125I-BHSP binding. After an exposure period of 10 days in darkness, the emulsion
dipped microautoradiograms were developed and
fixed as described. Corresponding sections were counterstained and examined under a light microscope.
Light- and dark-field photomicrographs were taken
from the stained sections and silver grains, respectively.
Data Analysis
Film macroautoradiograms were analyzed by computer-based densitometry. The optical density of the
autoradiogram was quantified by means of an image
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1991
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
slices of several layers of polymer containing a range
of increasing 1251 concentrations. Since tissue sections 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
subtracting nonspecific binding (obtained in the presence of 10 iiM of unlabeled SP) from total binding.
Classic computer analysis was used for the biochemical determination of the binding parameters (dissociation constant [Kd] and concentrations which inhibit
50% of the specific binding [IC50]) on rat retina sections. The kinetics of I25I-BHSP binding was assumed
to follow a bimolecular association model (secondorder kinetics).
Results
Characterization of 125 I-BHSP Binding
Kinetic analysis done by densitometry on rat retinal sections indicated that the association of 125I8O -
5O
TOO
15O
Time(min)
Fig. 1. Association and dissociation of I25I-BHSP binding to rat
retina sections. Retina sections were incubated with 65 pM 125IBHSP at room temperature for various times. The curve represents
specific binding that was calculated as the difference between binding in the presence and absence of 10-^M unlabeled substance P
(SP). Dissociation was initiated by the addition of 10-juM unlabeled
SP at 120 min, a time at which the association of the ligand had
reached the equilibrium. Values are given as the mean of triplicate
determinations in a typical experiment.
o
00
00
11
10
9
8
SP concentration
7
6
Log.M
Fig. 2. Competition between 125I-BHSP and unlabeled SP for
binding to rat retina sections. 65 pM 125I-BHSP were incubated
alone or in the presence of increasing concentrations of unlabeled
peptide SP at room temperature for 90 min. 125I-BHSP binding is
expressed as a percent of total binding. Values are given as the mean
of triplicate determinations.
BHSP resulted in a time-dependent increase in binding (Fig. 1). At room temperature, specific binding
reached a plateau in approximately 75 min; nonspecific binding was not significantly increased. About
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 125IBHSP 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
to 10~6 M), IC50 values were 0.17 nM. Curve analysis
obtained (obtained by regression lines computerized
by means of the least-squares method) indicated that
125
I-BHSP bound to high-affinity binding sites in
rat 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 in
attempt to establish the type of site identified by I25IBHSP in the rat retina. The order of potency for inhibition of binding was SP > NKA > NKB (Fig. 3, Table 1). A similar ranking was obtained when rabbit
eye sections were incubated with the tachykinins at
concentrations in the range of 10"8 to 10~5 M (data
not shown). Displacement curves showed that specific
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OCULAR 5UDSTANCE P BINDING SITES / Denis er al
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.SP
ASP
4-11
• NKA
0SP
l-4
ANKB
11
10
9
8
7
6
Peptide concentration -Log M
Fig. 3. Pharmacology of 125I-BHSP binding. 65 pM 125I-BHSP
were incubated alone or in the presence of increasing concentrations of unlabeled peptides at room temperature for 90 min. I25IBHSP binding is expressed as a percent of initial binding. Values are
given as the mean of four determinations.
binding of I25I-BHSP was strongly inhibited by
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,
respectively). On the other hand, senktide and septide
was ineffective, and S P ^ , NKB, and spantide had
some potency to inhibit retinal I25I-BHSP binding
(Table 1).
Autoradiographic Experiments
Representative autoradiograms of 125I-BHSP binding sites are shown in Figure 4-6. In the rat anterior
segment, SP binding sites were found in the iris,
mainly in the sphincter region (Fig. 4, Panel a). Analysis of histologic sections confirmed that labeling on
the pupil margin coincided with the iris muscular tissue. The density of silver grains was reduced dramatically in the presence of 10 IJM unlabeled SP (Fig. 4,
Panel b), indicating specific labeling in that structure.
In contrast, moderate 125I-BHSP binding densities
were noted over the other iridial structures and in the
corneal epithelium. No labeling occurred in structures such as the sclera and the ciliary processes. Binding seen on the lens fibers was not considered to be
specific since SP did not displace 125I-BHSP labeling.
In the posterior segment, the retina showed intensive
labeling; low concentrations of binding were noted in
the choroid. Binding sites were not uniformly distributed throughout the retina since a slight decrease of
binding at the ora serrata was observed. Autoradiograms of the emulsion-dipped slides provided detailed
histologic resolution of the retinal SP binding sites
(Fig. 5). Silver grains were localized primarily in the
inner 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 other
regions, particularly in the outer retina (outer nuclear
layer and photoreceptor cell layer). Similarly, very
low, but significant, concentrations of SP binding
sites were seen in the choroidal tissue. Some scleral
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 association with the iridial sphincter muscle; no labeling was
found either in the cornea, iris stroma, or ciliary body.
Low binding was also identified in the choroid. As in
the rat, lens fibers gave important nonspecific binding, ie, not displaceable in the presence of an excess of
unlabeled peptide. The retina also had high densities
of specific SP binding sites, mainly concentrated in
the IPL and GCL, as described in the rat.
Discussion
These results demonstrate that several structures
from albino rat and rabbit eyes (mainly the retina and
the iris sphincter muscle) have the capacity to bind
specifically a derivative analogue of SP, I25I-BHSP.
The characteristics of I25I-BHSP binding in the rat
retina strongly suggest that the ligand binds to SP receptors.
The apparent Kd of the high-affinity (0.13 nM)
binding site identified in this study was similar to
those reported in rat retina homogenates (0.2 nM),19
in rabbit optic sections (0.54 nM),28 and in human
retina (0.27 nM).29 Recent studies have described the
existence of multiple tachykinin receptors in the rat
retina.23 The agent, SP, belongs to a group of closely
Table 1.
Peptides
ICso (nM)
Substance P
Substance P^,,
Neurokinin A
Substance P,_4
Neurokinin B
Spantide
Substance P methyl ester
Septide
Senktide
0.17 ±0.02
3.39 ± 0.40
8.04 ± 0.57
35 ±4.34
52.8 ± 4.06
53 ± 1.53
54 ± 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 were
incubated for 90 min at room temperature with 65 pM I251-BHSP and increasing concentrations (10"" to 10~6 M) of unlabeled peptides. Nonspecific
binding of the radioligand was determined in the presence of 10-MM unlabeled SP. IC50 is defined as the molar concentration of the tested drug that
displaced 50% of the specific binding of I25I-BHSP on retinae sections. Autoradiograms were analyzed by computer-based densitometry with an image analyser. Values are given as the mean of triplicate determinations.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1991
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ISM
/|SM
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
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
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 micromolar 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.
related peptides known as tachykinins, all of which
contain a common C-terminal amino acid sequence:
Phe-X-Gly-Leu-Met-NH2.30 In 1983, two additional
tachykinins, NKA and NKB, were isolated in mammalian tissues.31 To date, three distinct receptors for
Vol. 02
B
Fig. 5. Microautoradiograms of I25I-BHSP binding in albino rat
posterior segment. Dark field (A) and bright field (B) were obtained
after incubation of 65 pM '"l-BHSP alone. Silver grains visualized
in the retina and choroid indicated specific SP binding sites since
labeling disappeared after incubation with an excess of unlabeled
peptide (not shown). GCL, ganglion cell layer; IPL, inner plexiform
layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL,
outer nuclear layer; SV, scleral vessel. Magnification X500.
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No. 6
OCULAR SUBSTANCE P BINDING SITES / Denis er ol
ISM
B
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
unlabeled SP (B). Auloradiograms were generated by apposition to
3
H-Hyperfilm for 7 days. (C) Histological section, C, cornea; ISM,
iris sphincter muscle; CP, ciliary processes; L, lens. Magnification
X3,
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
uvea originating from the trigeminal ganglion, 3334
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
from sensory nerves has also been shown to induce
marked inflammatory effects in cat39 and rabbit 40
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 concerning 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
Table 2.
Specific l2iI-BHSP bound
(fmol/mg tissue equivalent)
Area
Cornea
Iris stroma
Iris sphincter muscle
site. Furthermore, as described for SP binding sites in
other tissues, l9'25>26 affinity is encoded in the carboxy
terminal of SP since SP,_, j and SP4_n had the greatest
potency in inhibiting I25I-BHSP binding.
Autoradiographic visualization of 125I-BHSP binding sites in the anterior segment of the eye shows a
selective pattern of distribution. In both species, SP
1899
rat
rat
rabbit
rat
Retina
rabbit
rat
0.12
0.11
2.93
0.17
0.95
0.63
±0.01
±0.01
± 0.42
±0.01
±0.17
± 0.07
Regional distribution of specific BHSP binding sites in the albino rat and
rabbit eye. Results were obtained by transformation of optical densities as
described in the text to fmol/mg tissue equivalent ± SEM and were obtained
from at least 10 determinations throughout the area under examination.
Nonspecific binding was subtracted from each total value, so that data are
expressed as specific binding.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / May 1991
on the iris muscle through specific receptors. No binding sites was found in dilator or ciliary muscles, corroborating the fact that SP has no contractile effect on
these tissues.3843 One of the most interesting findings
of our study was the absence of high-affinity binding
sites in the rabbit iris and ciliary epithelia, known to
be the major site of the blood-aqueous barrier. It is
unlikely that the lack of binding sites in rabbit ciliary
body may be due to occupied receptor sites since sections were washed in a preincubation medium for 15
min before incubation with 125I-BHSP. Nevertheless,
we cannot exclude the possibility of the presence of
lower-affinity receptors or other unknown subtypes in
ciliary process. Such sites may be absent or not detectable by our autoradiographic procedures. However,
these results suggest that, in the rabbit, the peptide is
not directly involved in the blood-aqueous barrier
disruption observed in ocular neurogenic inflammation, as suggested by its weak and inconsistent effect.
In addition, we cannot exclude the possibility that SP
exerts some inflammatory actions in the eye through
an indirect mechanism; it has been demonstrated that
SP has a wide spectrum of inflammatory properties,
in particular mast cell degranulation with release of
histamine44 and arachidonic acid45 (and consequently
prostaglandin biosynthesis). Finally, the involvement
of SP in ocular neurogenic inflammation cannot be
generalized since it has been reported that the ocular
response to SP was species -dependent (cat, baboon,
or human iris are relatively insensitive to SP46). This
species variation is well illustrated in the our study by
the fact that the overall density of iridial SP binding
sites was higher in the rabbit than in the rat.
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 binding sites, such as the iris sphincter muscle, have been
reported previously to have the highest amounts of SP
immunoreactivity.47 In contrast, regions such as the
ciliary processes or the iridial stroma, also known to
contain SP immunoreactivity, did not have detectable SP binding sites. The existence of discrepancies
between the localization of receptors and the distribution of fibers or neurotransmitters has been clearly
demonstrated for SP in the brain.48
The SP binding activity in the scleral vessels (Fig. 5)
may be associated with the endothelium or vascular
constituents. This may be attributed to the chemotactic peptide, f-Met-Leu-Phe (fMLP)49 or the phagocytosis promoting peptide, tuftsin,50 which share structural similarities with SP. Furthermore, SP has been
shown to interact with the tuftsin receptors present on
the macrophage and polymorphonuclear leukocyte
plasma membrane.50 However, under our experimen-
Vol. 32
tal conditions, competition studies with increasing
concentrations of tuftsin (10~4 to 10~7 M) showed that
125
I-BHSP binding was not inhibited by tuftsin (data
not shown).
Although SP-containing neurons have been identified in the retina of mammals,51 including humans,52
very little is known about their functional role in this
tissue. These neurons are present in most species in
amacrine cells whose somata are located at the border
of the INL and IPL (which send processes primarily to
the IPL). Evidence for SP localization to ganglion
cells has also been demonstrated in some species, particularly rabbits.53 The localization of SP binding sites
we found was similar to the pattern of SP-immunoreactive cells previously described for this region. Neonatal monosodium glutamate treatment of rats
(which induces degeneration of the inner layers of the
retina) has been reported to cause a marked reduction
in 125I-BHSP retinal binding.19 Our autoradiographic
findings also agreed with this experiment since most
of the SP binding sites identified our study were concentrated in the IPL and GCL. Several observations
suggest a neuromodulator role for SP in retinal functions; it has been found to elicit 3H-dopamine release
from rabbit retinas18 and to exert excitatory effects on
carp cholinergic-sensitive ganglion cells activity.17 It
has been also shown to stimulate the accumulation of
inositol triphosphates in rabbit retinal cultures.54 The
presence of SP binding sites in the inner retina is an
additional indication that the peptide probably has
some regulatory action in visual processing.
Key words: substance P, substance P receptor, autoradiography, eye
Acknowledgments
The authors thank Odile Flamand and Yves Issoulie for
technical assistance.
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4. Von Euler US and Gaddum JH: An unidentified depressor
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