Download Localization of the prostaglandin F2 alpha receptor

Localization of the Prostaglandin F2a Receptor Messenger
RNA and Protein in the Cynomolgus Monkey Eye
Anette Ocklind* Staff an Lake,-\ Parti Wentzel,* Monica Nister,%
andjohan Stjernschantz*
Purpose. To determine the distribution of the prostaglandin F2a (FP) receptor within the
monkey eye.
Methods. The expression and localization of the FP receptor was studied by in situ hybridization
and immunohistochemistry. Cryosections of the eye were hybridized with a 35S-labeled FP
receptor riboprobe, and paraffin sections were immunostained with polyclonal antibodies
against an FP receptor peptide. Reverse transcriptase-polymerase chain reaction (RT-PCR)
was performed on cultured cell populations from the eye.
Results. Results of the three methods largely correlated with each other. The highest expression
of FP receptor mRNA and protein was found in the corneal, conjunctival, and iridial epithelium, the ciliary muscle, and ciliary processes. Iridial and choroidal melanocytes, the retina,
and the optic nerve expressed lower levels of both FP receptor message and protein. Using
immunohistochemistry, the FP receptor protein was found in connective tissue fibroblasts,
the corneal endothelium, and the vasculature; however, FP receptor expression was not detected using in situ hybridization. Witfi RT-PCR, cultured retinal pigment epithelial and
ciliary muscle cells from the cynomolgus monkey eye were found to express the FP receptor.
Conclusions. The FP receptor was found to be distributed widely in the ocular tissues, suggesting
an array of autocrine and paracrine functions of PGF2a in the eye. Invest Ophthalmol Vis Sci.
1996;37:7l6-726.
A rostaglandin (PG) F 2a is synthesized in several
ocular tissues in a variety of species, including humans. 1 2 The pharmacologic effects of PGF 2a are
species dependent and include, for example, contraction or relaxation of smooth muscle and a decrease in intraocular pressure. 3 " 5 The decrease in
intraocular pressure by topically administered
PGF 2a and PGF 2a analogs in primates and humans
has been shown to be mediated mainly by an increase in the uveoscleral outflow. 6 " 8 The physiologi-
From *Glaucoma Research laboratories, Pharmacia AB (publ), Uppsala;
fPreclinical R&D, Biopliarmaceuticals, Pharmacia AB (publ), Stockholm; and the
%Department of Pathology, University Hospital, Uppsala, Sweden.
Suf>ported by Pharmacia AB (publ), Uppsala, Sweden.
Presented in part at the 9th International Symposium on The OcuUir Effects of
Prostaglandins and Other Eicosanoids and at the annual meeting of the Association
for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, both in May
1995.
Submitted for publication April 17, 1995; revised December 28, 1995; accepted
January 3, 1996.
Proprietary interest category: E.
Reprint requests: Anette Ocklind, Glaucoma Research, Ijiboratories, Pharmacia AB
(publ), Pharmacia Pharmaceuticals, Uppsala S-751 82, Siueden.
716
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cal effect of PGF 2a appears to be mediated primarily
by the PGF 2a (FP) receptor. 9 1 0
The FP receptor cDNA from mouse, human, and
rat was recendy cloned, and a high nucleotide
sequence homology between the species was
found."" 14 The FP receptor is a member of the seven
transmembrane-receptor family and is linked to a guanosine triphosphate-binding protein. Stimulation of
the receptor induces intracellular signaling by phospholipase C-mediated phosphoinositide turnover,
which leads to the generation of the second messengers, inositoltriphosphate and diacylglycerol.15
The distribution of the FP receptor in the eye is
not well known. To understand and evaluate better
the effects of PGF2a and analogs in the eye, we have
studied the distribution of the FP receptor mRNA and
protein in ocular tissues by in situ hybridization and
immunohistochemistry. We have also used reverse
transcriptase polymerase chain reaction (RT-PCR) to
evaluate the expression of the FP receptor mRNA in
cultured cells originating from the monkey eye.
Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
Copyright © Association for Research in Vision and Ophthalmology
Localization of the Prostaglandin F2« Receptor
717
TABLE l.
Prostaglandin F2a Receptor-specific Primers Corresponding
to Positions 57-79 (Primer 1), 569-590 (Primer 2), and 547-568
(Secondary Primer 2) of the Human cDNA
Primer
Codon
Primary primer 1
Primary primer 2
Secondary primer 2
CAC AAG CTG CCA GAC GGA AAA C
CCA GTC TTT GAT GTC TTC TGT G
TTG TAG AAA CAC CAG GTC CTC G used together
with primary primer 1
MATERIALS AND METHODS
Animals
All animals involved in this study were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
son, WI) essentially according to the manufacturer's
instructions, with the exception that the labeling was
performed for 75 minutes at 37°C. Approximately
70% to 90% of the 35S-UTP was incorporated, to yield
a final specific activity of 1 X 109 cpm/fig RNA.
In Situ Hybridization
For in situ hybridization, cryosections were rehydrated
Four adult cynomolgus monkeys (Macaca fascicularis) and permeabilized in 0.2% Triton X-100 (Life Techwere anesthetized with a mixture of ketamine (20 mg/
nologies, Louisville, IL) in PBS, washed in PBS, dikg body weight) and xylazine (1.5 mg/kg body weight)
gested with Proteinase K (Boehringer Mannheim,
and perfused intracardially with 4% neutral buffered
Mannheim, Germany; 1 fxg/ml in 10 mM Tris-HCl
paraformaldehyde. For in situ hybridization, the eyes
with 5 mM ethylenediaminetetraacetic acid) for 15
were enucleated and bisected equatorially, the lens
minutes at 37°C, treated with 0.1 M glycine in PBS,
and vitreous were discarded, and the hemispheres
postfixed in 4% paraformaldehyde for 3 minutes, and
were further fixed for 4 hours at 4°C. After extensive
rinsed in PBS. Sections were acetylated in 0.25% (vol/
washing in 15% sucrose in phosphate-buffered saline
vol) acetic anhydride with 0.1 M triethanolamine, pH
(PBS), pH 7.4, the anterior and posterior segments
8, for 10 minutes, rinsed in distilled water, and dried.
were subdivided into sectors. Tissue samples in OCT
Hybridization was performed in 50% formamide, 10%
(Miles, Elkhart, IN), were frozen in liquid nitrogendextrane sulfate, 5 X Denhardt's solution, 10 mM vacooled isopentane and stored at — 80°C for less than nadyl ribonucleoside, 100 /ig/ml denatured salmon2 weeks. Tissues were cryosectioned at 4 to 7 fim,
sperm DNA, 10 fiM dithiothreitol (DTT), and 5 X 108
mounted on Superfrost Plus slides (Gerhard Menzel
cpm probe/[A. Twenty microliters of the hybridization
GmbH KB, Braunschweig, Germany), refrozen at
mixture was applied to each tissue section, which was
—80°C, and analyzed within 1 month. For immunohis- coverslipped and incubated for 4 hours at 48°C. Slides
tochemistry, enucleated eyes were further fixed for 7
were rinsed in five changes of 2 X 0.15 M NaCl and
hours at room temperature. After 4 hours, a calotte
2 X 15 mM sodium citrate (2 X SSC) with 0.1% (vol/
was cut from each eye to facilitate the penetration of
vol) sodium dodecyl sulfate and 10 mM DTT at room
the fixative. After washing in PBS overnight at 4°C,
temperature and in four changes of 0.1 X SSC/0.1%
the eyes were paraffin embedded and sectioned at 4
sodium dodecyl sulfate per 10 mM DTT at 48°C, furto 5 /im according to standard methods.
ther rinsed in 2 X SSC, treated with 10 /Ltg/ml RNase
in 2 X SSC for 15 minutes at 37°C, and finally rinsed
Preparation of Probes
in 2 X SSC. Tissue sections were dehydrated in ethanol
with
0.3 M ammonium acetate and were air dried overRiboprobes were synthesized from PCRII (Invitrogen,
night.
Hybridized preparations were autoradioSan Diego, CA) plasmid vectors containing dual SP6
graphed
with NTB-2 emulsion (Eastman Kodak, Rochand T7 promoters and nucleotides 56 to 566 of the
NY)
diluted 1:1 in distilled water. After exposure
ester,
coding region of the human FP receptor, that is, the
14
11
to 14 days at 4°C, the slides were developed
during
transmembrane regions 1 to 4 (see Fig. 4).
in
Dektol
(Kodak),
counterstained with hematoxylinAntisense and sense probes were transcribed from
eosin,
mounted,
and
examined with combined epipoplasmids linearized with BamHl using an RNA tranlarization
and
bright-field
microscopy.
scription kit (Ambion MAXI Script, Austin, Texas)
35
35
in the presence of S-UTP (uridine 5'-[ S]thioCell Cultures
triphosphate; Amersham, Solna, Sweden). The transcription reaction was carried out with SP6 (antisense)
Cynomolgus monkey retinal pigment epithelial cells
and T7 (sense) polymerases (Promega Biotech, Madiwere isolated from four freshly enucleated eyes essenTissue Preparation
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Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
FIGURE l. In situ hybridization of cynomolgus monkey eye tissues with 35S-labeled prostaglandin Fa,f (FP) receptor riboprobes. Photomicrographs showing in situ hybridization with
antisense riboprobe (left panels) and sense riboprobe (right panels). The hybridization signal
obtained with the sense probe is very low. (A,B) Anterior cornea. Corneal epithelium shows
specific hybridization. (C,D) Longitudinal portion of ciliary muscle. (E,F) Ciliary process.
Specific hybridization is shown on both the nonpigmented (arrow) and pigmented (arroxvhead) epithelium. <G,H) Iris. FP receptor mRNA is visualized on the pigment epithelium
(pe) and iris stromal melanocytes (arrow). (IJ) Inner retina. Specific hybrids are shown on
the ganglion cell (gc) layer. (K,L) Outer retina. FP receptor mRNA is shown on retinal
pigment epidielium (arrow), inner segments, and outer nuclear layer of photoreceptors
(on). (M,N) Cross-section of optic nerve fiber bundles exhibiting specific hybridization.
Photomicrographs are taken in simultaneous epipolarized and bright-field illumination.
Magnification, X400.
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Localization of the Prostaglandin F2a Receptor
FIGURE l. (Continued)
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Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5
FIGURE 2. Fluorescence photomicrographs of cynomolgus monkey eye sections stained with
antibodies to the human prostaglandin F2a (FP) receptor. (A) FP receptor immunoreaction
is localized to the corneal epidielium and, to a lesser extent, to the stromal fibroblasts
(arrow). (B) Preabsorbed antibodies block the immunoreaction, as shown in the cornea.
(C) Preimmune antibodies show little binding, as shown in the cornea. (D) Corneal endothelium and (E) lens epithelium are immunopositive. (F) The entire ciliary muscle is iramunopositive, as shown in the longitudinal muscle portion. (G) Positive immunoreaction is seen
in the ciliary processes (arrow). (H) In the retina, the outer nuclear (on) and inner nuclear
(in) layers, as well as the ganglion cell (gc) layer, show positive immunoreaction. For the
retinal pigment epithelium, see Figure 3. (I) Immunoreactive optic nerve fiber bundles are
in longitudinal section visualized as four broad horizontal bands next to lamina cribrosa
(lc). (J) Blood vessels (arrow), as exemplified in the limbal region, are immunopositive. (K)
The lateral rectus extraocular muscle (cross-section through muscle bundles) is immunopositive. Magnification, X200.
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Localization of the Prostaglandin F2tr Receptor
FIGURE 2.
ffl
(Continued)
tially according to a previously published method."1
Two animals were killed by an intracardiac injection
of pentobarbital (100 mg/kg body weight) under ketamine (20 mg/kg body weight) and xylazine (1.5 mg/
kg body weight) anesthesia, and the eyes were enucleated. Each eye was bisected along the equator, and
the vitreous was removed. The posterior eye cup was
rinsed in PBS, pH 7.4, supplemented with 25 mM
Hepes and 1% antibiotic-antimycotic solution (Life
Technologies, Paisley, Scotland), and the neural retina was removed gently. After rinsing, the retinal pigment epithelial cells were released and collected from
Bruch's membrane by gently pipetting several changes
of the rinsing solution in the eye cup. Cells were cultured in equal concentrations of Dulbecco's modified
Eagle medium (DMEM) and Ham's F-.12 supplemented with 15% fetal calf serum (FCS), 2 mM Lglutamine, 50 //g/ml gentamicin (all from Life Technologies), and 1 ng/ml human recombinant basic fibroblast growth factor (Sigma Chemical, St. Louis,
MO) for approximately 1 week at 37°C, 5% COi> humidified air, after which the concentration of FCS was
reduced to 10%. Confluent cultures were subcultured
by trypsinization. In confluent cultures without basic
fibroblast growth factor, the identity of the retinal pigment epithelial cells was confirmed by their flat cuboi-
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dal appearance and by the presence of pigment granules. Immunocytochemical staining with monoclonal
antibodies (Clone KL 1; Immunotech, Marseilles,
France) against the cytokeratins 1, 2, 5, 6, 7, 8, 11, 14,
16, 17, and 18 always was found in more than 95% of
the cells in culture. Cultured human retinal pigment
epithelial cells are reported to express the cytokeratins
7, 8, 18, and 19.17
Cynomolgus monkey ciliary muscle cells were isolated and cultured from four freshly enucleated eyes
essentially according to a previously published
method18 with some adaptations. Briefly, each eye was
divided by cutting between the ora serrata and the
equator, and the lens and the iris were removed. The
pars plana region of the ciliary body was cut out and
divided radially into approximately four segments.
The segments were incubated in 2.4 U/ml dispase
(Boehringer Mannheim) in DMEM supplemented
with 10% FCS for approximately 15 minutes at 37°C,
5% CO2, air, after which the epithelial layers and vessels on the posterior side and choroidal melanocytes
on the anterior side were scraped from the ciliary muscle region. Originating from the longitudinal and radial ciliary muscle, cells were dissociated by cutting
the tissue into 1-mm3 pieces and incubating in 0.1%
collagenase (Sigma) in DMEM supplemented with
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Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
10% FCS for 2 hours at 37°C. Cells were collected and
cultured in the medium used for the retinal pigment
epithelial cells with the further addition of 10 ng/ml
human recombinant platelet-derived growth factor BB
(Sigma). At confluence, the cells were subcultured by
trypsinization. In confluent cultures without growth
factors, the identity of the ciliary smooth muscle cells
was apparent by their "hill-and-valley" growth pattern.1'1 Immunocytochemically, more than 95% of the
cells always stained brightly for smooth muscle a-actin
(antibodies from Sigma).
Reverse Transcriptase—Polymerase Chain
Reaction
Specific primers were designed for the human FP receptor as presented in Table 1. The design of the
primers was based primarily on the more conserved
homology between species in the transmembrane regions and in certain regions conserved in the family
of seven transmembrane receptors—in this case, the
tryptophan, cysteine, phenylalanine motif present in
most, if not all, seven transmembrane receptors. From
a set of combinations of primers in these regions, the
presented primers were shown to hybridize and amplify cynomolgus monkey FP cDNA. RT-PCR was performed on mRNA isolated from cell cultures of cynomolgus monkey retinal pigment epithelial and ciliary
muscle cells. The RT-PCR products were analyzed
on agarose gel, and bands of the expected sizes were
cloned and sequenced. Deduced amino acid sequences were analyzed for homology to the human
FP receptor sequences.
For the experiments, cells at passages 2 to 4 were
grown to confluence and harvested. mRNA was isolated using Dynals mRNA Direct system (Dynal A/S;
Oslo, Norway) according to the manufacturer's recommendations. Approximately 500,000 cells of each
finite cell line were used in the enrichment, where
mRNA is covalently bound to an oligo-dT labeled Dynabead. Using reverse transcriptase, the first-strand
cDNA was synthesized directly on the Dynabead with
the oligo-dT as a reverse transcriptase primer. The
second-strand cDNA was synthesized using a known
3' sequence primer from the prostaglandin receptor,
resulting in double-stranded cDNA. This doublestranded cDNA was amplified with receptor-specific
nested primers for PCR-amplifying cDNA, according
to the manufacturer's recommendations. The FP receptor PCR reactions were performed in 50 //I final
volume, including 5% dimethyl sulfoxide, 200 /xM deoxyribonucleotides, and 20 pmol of each primer.
The PCR products from these reactions were analyzed on a 0.9% low melt preparative grade agarose
gel (BioRad Laboratories, Melville, NY). DNA fragments of the expected size were thymidine-adenine
cloned according the manufacturer's recommenda-
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tions (PCR II; Invitrogen) and sequenced on an Applied Biomodel 373A DNA sequencing system (Applied Biosystems, Foster City, CA) according to the
manufacturer's protocol for their Taq Dye Dioxy terminator cycle sequencing kit. The generated data
were processed on a VAX computer using sequence
analysis programs (Genetics Computer Group, Madison, WI).20
Immunohistochemistry
A polyclonal antiserum against a synthesized peptide
corresponding to the first extracellular loop (that is,
between transmembrane regions 2 and 3) of the FP
receptor was raised in rabbits and the immunoglobulin (Ig) G fraction was used. Specificity of the antibody
was determined using stable FP receptor-transfected
versus nontransfected host cells and by competition
with its corresponding antigen. Specificity also was
seen on Western blots of yeast-expressed FP receptor
protein (data not shown). Preimmune serum was collected before immunization, and the immunoglobulin
fraction was isolated.
Indirect immunofluorescence staining was performed on paraffin sections as previously described.21
Briefly, antigenic sites were exposed by incubating the
sections in 0.1% trypsin (DAKO, Glostrup, Denmark)
in 0.1% CaCLj for 5 minutes at 37°C. Autofluorescence
and nonspecific binding were blocked with 0.15 M
ethanolamine, pH 7.5. Nonspecific binding were
blocked further with 5% normal goat serum, and possible endogenous biotin was blocked with avidin-biotin blocking solutions (DAKO). Sections were incubated with immune and preimmune antibodies at 4
//g/ml for 90 minutes and then with biotinylated goatantirabbit IgG (1:250; Sigma) for 30 minutes. Binding
sites were visualized with streptavidin-labeled Texas
red (1:100; Amersham) and examined by epifluorescence microscopy.
In some experiments, gold-labeled goat anti-rabbit IgG (1:40; Amersham) were used instead of the
biotinylated secondary antibodies. These sections
were treated with Silver IntenSE (Amersham), counterstained with hematoxylin, and examined by combined epipolarization and bright-field microscopy.
Control experiments were performed using preimmune IgG or PBS in place of the primary antibodies.
In addition, control sections of all experimental tissues were incubated with primary antibodies that had
been preabsorbed with the FP receptor peptide and
the conjugation protein used for the immunization.
For the preabsorption experiments, antibodies were
mixed with the peptide and its conjugation protein at
a protein concentration ratio of 10:1, and the mixture
was incubated for 30 minutes at 37°C. After centrifugation for 4 minutes at 10,000g, the supernatant was
collected and used for the staining.
723
Localization of the Prostaglandin F2a Receptor
TABLE 2.
Relative Expression of FP Receptor
mRNA and of Immunoreactive FP Receptor
in the Monkey Eye
Tissue
Conjunctiva
Epithelium
Stroma
Cornea
Epithelium
Stroma
Endothelium
Iris
Epithelium
Melanocytes
Sphincter
Lens
Epithelium
Ciliary body
Ciliary muscle
Ciliary processes
Trabecular meshwork
Choroid
Melanocytes
Retina
Ganglion cells
Photoreceptors
Inner nuclear layer
Pigment epithelium
Optic nerve
Nerve fibers
Vessels
Arteries
Veins
Rectus muscle
ISH*
IFf
ND
mRNA on most cell types (Fig. 1, left panels), as detected by comparison with the sense probe hybridization (Fig. 1, right panels). The highest accumulations
of silver grains were found throughout the corneal
(Fig. 1A) and conjunctival epithelium, ciliary muscle
(Fig. 1C), ciliary processes (Fig. IE), iris pigment epithelium (Fig. 1G), choroidal melanocytes, and photoreceptors of the retina (Fig. IK). Within these cell
populations, the hybridization signals were homogeneously distributed with equal intensities. In the corneal epithelium (Fig. 1A), conjunctival epithelium,
and iridial pigment epithelium (Fig. 1G), FP receptor
mRNA appeared to be expressed in all cell layers. In
the ciliary muscle (Fig. 1C), specific hybridization was
seen in longitudinal, radial, and circular muscle portions. In the ciliary processes (Fig. IE), FP receptor
mRNA was shown on both epithelial cell layers, although the nonpigmented epithelium was less positive. In the photoreceptor layer (Fig. IK), FP receptor
mRNA was localized to the outer nuclear layer and
the inner segments of rods and cones. Weak specific
hybridization was found in the iridial melanocytes
(Fig. 1G), iris sphincter muscle, trabecular meshwork,
optic nerve fibers (Fig. 1M), retinal pigment epithelial
cells (Fig. IK), retinal inner nuclear layer and ganglion cell layer (Fig. II). In the optic nerve, most silver
grains were seen to overlie the nerve fibers (Fig. 1M).
No specific hybridization signal was detected in the
corneal (Fig. 1A) and conjunctival fibroblasts, corneal
endothelium, or vessels. The lens epithelium was not
studied because the lens was removed before the cryopreservation.
ND
* ISH = in situ hybridization. Hybridization is monitored as: — =
no signal above background; + = weak positive signal; and + +
= strong positive signal. ND = not determined,
f IF = immunofluorescence. Immunoreactivity is monotored as:
— = no signal; + = weak positive signal; and ++ = strong
positive signal.
RESULTS
In Situ Hybridization
In situ hybridization results are shown in Figure 1
and are summarized in Table 2. Control hybridization
(Fig. 1, right panels) with sense probe exhibited almost no labeling, as indicated by the sparse distribution of silver grains. However, pigmented cells and
retinal nuclear layers generally displayed a slightly
higher background level of silver grains than other
cells. When the in situ hybridization procedure was
performed without probe, no silver grains were seen
on any cells (not shown).
In situ hybridization with the FP receptor antisense probe revealed a low, but specific, expression of
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Inimunohistochemistry
Immunohistochemistry results are shown in Figures 2
and 3 and are summarized in Table 2. Indirect immunofluorescence staining with the polyclonal antibodies showed the FP receptor to be present in small
amounts in all ocular tissues examined (Fig. 2). Preabsorbed antibodies reduced the signal intensity to near
background levels (Fig. 2B). Preimmune serum
showed little binding (Fig. 2C). The low nonspecific
binding was achieved only by soaking the fixed specimens overnight in PBS to remove excess formaldehyde, by examination of the unreacted formaldehyde
groups in ethanolamine, and by brief washings in PBS
containing Triton X-100. Autofluorescence that could
not be quenched occurred in the retinal pigment epithelium and in iridial melanocytes. Consequently, indirect immunogold staining and silver enhancement
was used as a complimentary method to visualize binding sites in pigmented cells. The FP receptor protein
was found predominantly in the conjunctival and corneal epithelium (Fig. 2A), notably in the basal layers,
ciliary muscle (Fig. 2F), ciliary processes (Fig. 2G),
iris pigment epithelium (Fig. 3C), lens epithelium
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Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
FIGURE 3. Photomicrographs of immunogold-silver detection of the human prostaglandin F2a (FP) receptor protein
in cynomolgus monkey eye tissues. (A, C) Primary FP receptor antibodies. (B, D) Control staining using preimmune
antibodies. (A) Immunoreactive retinal pigment epithelium
is seen as a white horizontal band. (B) Preimmune antibodies show litde binding. (C) Iridial melanocytes (arrow) and
pigment epithelium (pe, arrowhead) are immunopositive.
(D) Nonspecific binding of preimmune antibodies is low.
Photomicrographs were taken in simultaneous epipolarized
and bright-field illumination. Magnification, X400.
Reverse Transcriptase-Polymerase Chain
Reaction
Retinal pigment epithelial and ciliary muscle cells in
culture expressed the FP receptor, based on RT-PCR.
One band corresponding to the expected 465-bp fragment was found. Cloning and sequence analysis confirmed the identity to the FP receptor. At the amino
acid level, the homology between the cynomolgus and
the human FP receptor was found to be 99% in the
amplified TM1-TM4 region of the receptor (Fig. 4).
DISCUSSION
(Fig. 2E), and retinal pigment epithelium (Fig. 3A).
In the ciliary muscle, staining appeared throughout
all muscular portions. In the ciliary processes, most of
the immunoreaction was located to the nonpigmented epithelium. The FP receptor seemed least
abundant in fibroblasts from various connective tissues (Fig. 2A), corneal endothelium (Fig. 2D), iridial
melanocytes (Fig. 3C), retinal neural and photoreceptor layers (Fig. 2H), extraocular muscle (Fig. 2K), and
optic nerve (Fig. 21). Bloodvessels stained weakly. Occasionally, limbal vessels (Fig. 2J) showed more intense immunoreaction.
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The purpose of the study was to investigate the distribution of the FP receptor in the monkey eye. Results
obtained with an antiserum against the FP receptor
correlate with immunohistochemical studies21 of the
FP receptor in the rat eye in which the receptor was
distributed in various tissues in the eye and in the rest
of the body. Receptor binding as well as pharmacologic and molecular biologic studies have suggested
the presence of the FP receptor in various tissues of
the reproductive, renal, gastrointestinal, cardiovascular, digestive, respiratory, endocrine, and nervous systems1222"24 in various species. Our study on the distribution of the FP receptor in the monkey eye seems
to agree fairly well with published experiments25 on
human cadaver eyes using ligand-binding techniques,
in which the FP receptor was found in the ciliary muscle, iris pigment epithelium, iris sphincter muscle, and
retina.
Overall, our results indicate a low abundance of
FP receptor transcripts and protein. The signals were
low in most ocular tissues, as evidenced by the low
amount of silver grains despite an exposure time of 2
weeks. Longer exposure times increased the background level of grains and did not increase sensitivity.
Detection of the autoradiographic silver grains was
enhanced strongly with epipolarization microscopy.26
Under bright-field illumination, most grains were invisible. Furthermore, epipolarization microscopy permitted a clear-cut distinction between silver grains and
725
Localization of the Prostaglandin F2a Receptor
human
SVIFMTVGILSNSLAIAILMKAYQRFRQKSKASFLLLASGLVITDFFGHL
monkey
ISVIFMTVGILSNSLAIAILMKAYORFROKSKASFLLLASGLVITDFFGHL
lllllllllllllllllllllllllllllllllllllllllllllllll
TM1
FIGURE 4. Deduced amino
acid residue homology between human and cynomolgus monkey prostaglandin
¥2a receptors in the amplified polymerase chain reaction sequence. Transmembrane regions are indicated
as TM1-TM4 and correspond to nucleotides 56 to
566.
TM2
INGAIAVFVYASDKeWIRFDQSNVLCSIFGICMVFSGLCPLLLGSVMAIERCIGVTKPIF
INGAIAVFVYASDKdWIRFDQSNVLCSIFGICMVFSGLCPLLLGSVMAIERCIGVTKPIF
TM3
HSTKITSKHVKMMLSGVCLFAVFIALLPILGHRDYKIQASRTWCFY
HSTKITSKHVKMMLSGVCLFAVFIALLPILGHRDYKIQASRTWCFY
TM4
pigment granules derived from the pigmented cells.
Only metal particles such as silver grains deflect polarized light, which is visualized as brightly shining spots
against a dark background. 26 With this technique, the
signals were found to be specific. When the epipolarization was combined with bright-field illumination,
cell morphology was clearly visible.
Our immunohistochemical studies of the distribution of FP receptor protein yielded results largely in
agreement with our in situ studies. In addition, in
this assay, the signals were low. Immunofluorescence
studies required bright illumination, and photomicrography required long exposure times. In cell populations labeled by our antibodies, the strongest signals
for each of the two techniques correlated with each
other, except for the choroidal melanocytes and the
photoreceptor layer. In these cell layers, FP receptor
transcripts seemed to be relatively more abundant
than the FP receptor protein. Retinal pigment epithelial cells, retinal ganglion cells, and nerve fibers in the
optic nerve exhibited a slighdy higher-than-background level of silver grains. The low abundance of
the FP receptor in these tissues was, except for the
retinal epithelium, confirmed by the immunohistochemical staining.
Interestingly, FP receptor transcripts appeared to
be absent in vessels, corneal and conjunctival stroma,
and scleral and corneal endothelium, whereas the FP
receptor protein was detected in all these tissues. The
differences between mRNA and protein distributions
may be caused by different levels of sensitivity of the
two methods. The antiserum against the FP receptor
protein was produced against a small synthetic peptide
corresponding to die first extracellular loop of the
human FP receptor. Preabsorption experiments confirmed antibody specificity. Because of similarities between different PG receptors, it can be argued that
antiserum cross-reacts with other PG receptors. However, we chose this particular peptide for the genera-
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tion of antibodies because amino acid sequence differs
most between PG receptors of different types but is
well conserved between species.21'27 Cross-reactivity of
the riboprobe with other PG receptor mRNA seems
unlikely because of the considerable length of the
probe. The reported overall amino acid identity between human PG receptors is only 27% to 42%. 27 It
cannot be excluded that the relative abundance of
mRNA and protein might differ with respect to cell
type because of synthesis, degradation, or secretion of
protein in various cell types. In structures for which
we can detect colocalization of mRNA and receptor
protein, the FP receptor is present. If only receptor
protein can be detected, however, it cannot be excluded that the immunoreaction is nonspecific.
To confirm the results obtained with in situ hybridization and immunohistochemistry, we took advantage of the possibility of analyzing cultured cells
originating from the cynomolgus monkey eye. Here,
we used RT-PCR as a different method to analyze the
FP receptor mRNA. Taking into account the differentiation and possibly altered phenotype of cultured
cells when compared to cells of intact tissues, our R T PCR data showed good correlation with the results
obtained with tissue sections. The high amino acid
homology in the TM1-TM4 region implies that the
monkey is a good model for humans.
These FP receptor results raise questions about
die physiological role of PGF2a in the eye. Based on
the widespread ocular distribution of the FP receptor,
we can assume that, for a local hormone, PGF2a has
a great variety of functions in the eye. Because of this
widespread receptor distribution, we postulate die
presence of cell type-specific intracellular signals to
account for the many different biologic effects induced by PGF 2a .
Key Words
immunohistochemistry, in situ hybridization, localization,
monkey eye, prostaglandin F2a (FP) receptor
726
Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5
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