December 2001
Volume 42, Issue 13
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Glaucoma  |   December 2001
Detection of Prostaglandin EP1, EP2, and FP Receptor Subtypes in Human Sclera
Author Affiliations
  • Todd L. Anthony
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • James D. Lindsey
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • Makoto Aihara
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • Robert N. Weinreb
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
Investigative Ophthalmology & Visual Science December 2001, Vol.42, 3182-3186. doi:
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      Todd L. Anthony, James D. Lindsey, Makoto Aihara, Robert N. Weinreb; Detection of Prostaglandin EP1, EP2, and FP Receptor Subtypes in Human Sclera. Invest. Ophthalmol. Vis. Sci. 2001;42(13):3182-3186.

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Abstract

purpose. To examine the expression of five prostaglandin (PG) receptors, EP1, EP2, EP3, EP4, and FP and their corresponding mRNA transcripts in human sclera and cultured human scleral fibroblasts (HSFs).

methods. Primary cultures of HSFs were established from donor eyes. Also, sclera from human donor eyes was snap frozen and sectioned. Immunocytochemistry was performed on HSFs and tissue sections with subtype-specific antibodies to the EP1, EP2, EP3, EP4, and FP receptors. The presence of mRNA for the receptor subtypes was examined from total RNA obtained from human sclera and confirmed with restriction digest analysis.

results. Positive EP1 and FP receptor immunoreactivity was observed in fibroblasts within the sections from human sclera. In primary cultures of HSFs, EP1 and FP labeling was observed over the entire cell surface. EP2 immunoreactivity within HSFs was mostly present in the juxtanuclear region. RT-PCR analysis of total RNA isolated from human sclera and HSFs confirmed the presence of EP1, EP2, and FP receptor subtypes. The identity of the polymerase chain reaction products was confirmed by restriction enzyme analysis. No mRNA or immunoreactivity above basal levels was detected for the EP3 and EP4 prostanoid receptor subtypes in tissue sections or primary cultures.

conclusions. The EP1, EP2, and FP receptor subtypes are present in HSFs, suggesting that these cells may respond to endogenous PGs and their structural analogues through interaction with these receptor subtypes.

Prostaglandins (PGs) produce a wide range of cellular effects through the activation of specific membrane receptors. The classification of the prostanoid receptors has been defined by the relative potencies of the natural agonists and confirmed by molecular cloning studies leading to the identification of the EP1, EP2, EP3, EP4, DP, FP, and TP receptors. 1 In the eye, some PGs have been demonstrated to be effective ocular hypotensive agents in humans and other species. 2 The PGF analogue latanoprost is used widely to lower intraocular pressure (IOP) and treat human glaucoma. 3  
Although the precise mechanism for the IOP-lowering effect of latanoprost after topical application is not known, there is considerable evidence that supports the idea that PGF, and possibly latanoprost, lowers IOP by enhancing uveoscleral outflow. 4 5 6 7 It is possible that this is mediated by activation of prostanoid receptors in ciliary muscle and other tissues within the uveoscleral outflow pathway. However, our knowledge of the distribution of the prostanoid receptor subtypes in the eye is still limited. This paucity of knowledge prevents a complete understanding of the ocular effects of the PGs and their analogues. 
The uveoscleral outflow pathway consists of the iris root, the ciliary muscle cells, the supraciliary and suprachoroidal spaces, and the sclera. 8 The extent to which the sclera is involved in the uveoscleral outflow pathway is not known. Sclera is composed of collagen fibrils embedded within a proteoglycan matrix. The collagen and proteoglycans in the sclera are produced and maintained by fibroblasts. These fibroblasts may play an integral part in the regulation of the scleral extracellular matrix. To determine whether scleral fibroblasts might express receptors for endogenous or topically applied PGs, we examined the expression of the EP1, EP2, EP3, EP4, and FP receptor subtypes in human sclera and in cultured scleral fibroblasts. 
Materials and Methods
Human Sclera Tissue Preparation and Primary Culture
Eyes from two donors (aged 36 and 79 years; obtained from the San Diego Eye Bank) were enucleated within 5 hours of death and stored at 4°C. Donors had no known history of glaucoma or other eye diseases. All procedures in this study followed the University of California San Diego guidelines for use of human tissue in research. 
Connective tissue, blood vessels, muscle, and conjunctiva were dissected from the exterior surface of the globes. The anterior chamber was removed by equatorial incision approximately 4 to 5 mm behind the corneal limbus. After cutting the zonule, the lens was removed and the remaining anterior segment was embedded in optimal cutting temperature compound (OCT; Tissue-Tek, Miles, Inc., Elkhart, IN) and snap frozen by immersion in a dry-ice and ethanol bath. Sections 10 μm thick were cut using a cryostat and then placed on positive-charged microscope slides (OptiPlus; BioGenex, San Francisco, CA). 
Primary human scleral fibroblast (HSF) cultures were established from whole-tissue explants. Briefly, an incision was made (3–5 mm) anterior to the optic nerve head. Scleral pieces approximately 1 to 2 mm thick were placed uveal side down under sterile coverslips. Tissue segments were incubated in DMEM-F-12 medium supplemented with 10% fetal calf serum (FCS; Gemini, Calabasas, CA), 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 mg/ml amphotericin B (Fungizone; all antibiotics from Gibco BRL, Grand Island, NY) and 1 ng/ml basic fibroblast growth factor (R&D Systems, Minneapolis, MN). After 3 to 4 weeks, primary cells were passaged once to 75-cm2 flasks to establish the cell lines and then to 35-mm dishes for analysis. 
Isolation of RNA
Tissue fragments were homogenized in 10 ml of reagent (TRIzol; Gibco BRL; Polytron homogenizer; Brinkman Instruments, Westbury, NY). The homogenate was transferred to sterile 1.5-ml microcentrifuge tubes in 1-ml aliquots and incubated for 5 minutes at 25°C. Chloroform (200μ l) was added to each tube. The tubes were mixed by brief agitation and incubated for an additional 3 minutes at 25°C. The samples were then centrifuged (12,000g) for 15 minutes at 4°C, and the aqueous phase was carefully transferred to fresh sterile 1.5-ml microcentrifuge tubes. Isopropanol (500 μl/tube) was added and allowed to incubate for 10 minutes at 25°C to initiate RNA precipitation. Samples were centrifuged (12,000g) for 10 minutes at 4°C and the supernatant was removed. The RNA pellet was washed with 75% ethanol-diethylpyrocarbonate (DEPC)-treated water and air dried. RNA was resuspended in a total volume of 40 μl of DEPC-treated water, and quality was checked by gel electrophoresis. 
Reverse Transcription–Polymerase Chain Reaction
RT-PCR was performed essentially as previously described, 9 with total RNA isolated from human sclera. Primers were chosen to amplify unique regions within the individual human FP and EP receptor subtypes. All PCR primer pairs were 100% homologous with the reported cloned sequences of the human PG receptors (Table 1) . The PCR reaction mix (final volume, 50 μl) contained 5 μl of the RT reaction, 5 μl of 10× PCR buffer (Gibco BRL), 1 μl of 10 mM dNTP mixture, 1.5 μl of 50 mM MgCl2, 2.5μ l of the sense and antisense primers (20 μM), and 0.5 μl Taq polymerase (5 U/μl; Gibco BRL). The PCR program consisted of an initial step at 95°C for 3 minutes, followed by 30 cycles at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, and a final step at 72°C for 7 minutes. Products were analyzed by electrophoresis in 1% agarose gels. The human plasmids encoding the EP1, EP2, EP3, EP4, and FP receptors used for the positive controls of the RT-PCR reactions were a gift from John Regan (University of Arizona, Tucson, AZ). 
Immunofluorescence Microscopy
Primary antibodies to the human EP1, EP2, EP3, EP4, and FP receptors were generated using recombinant fusion proteins consisting of glutathione-S-transferase (GST) and a portion of the carboxyl terminus for the human EP3, EP4, and FP receptors or GST and the third intracellular loop of the human EP1 and EP2 receptors (also from John Regan). These antibodies have been characterized, using COS-7 cells transfected with plasmid DNA encoding each PG receptor subtype. 9 10 Secondary antibodies were as follows: goat anti-chicken (Alexa Fluor 568, 1:200 dilution; (Molecular Probes, Eugene, OR) for the EP antibodies and goat anti-rabbit (Alexa Fluor 568, 1:200 dilution; Molecular Probes) for the FP antibody. 
For immunolabeling, human sclera tissue (10-μm-thick anterior segments) were postfixed in 4% paraformaldehyde and phosphate-buffered saline (PBS) for 10 minutes at room temperature. Sections were washed once in PBS and then placed in 30 mM sodium chloride and 300 mM SSC buffer for 10 minutes at room temperature. Sections were placed in 100 mM glycine solution for 15 minutes, then washed in SSC buffer and permeabilized with SSC containing 0.1% Triton X-100 for 1 hour. After an overnight incubation at 4°C with the primary antibody (1:100–1:300 dilution), the sections were washed with SSC, incubated for 1 hour at room temperature with secondary antibody at a dilution of 1:1000, washed again, and mounted under coverslips for viewing. Before immunostaining, HSFs were grown on glass coverslips. The cells were fixed with 4% formaldehyde and PBS solution (freshly prepared from paraformaldehyde) for 5 minutes at room temperature. Labeling with the antibodies was performed as previously described. 
Results
Human PG Receptor Subtypes in Scleral Tissue
Tissue sections from human anterior segments (10 μm, n = 2 separate eyes) were labeled with antibodies to human EP1, EP2, EP3, EP4, and FP receptor subtypes, and scleral tissue was viewed by immunofluorescence microscopy. Figure 1 shows the fluorescence of human sclera after labeling with antibodies to the human EP1 and FP receptor subtypes. Strong immunoreactivity was observed in scleral fibroblasts after labeling with the EP1 receptor antibody (Fig. 1D) . Immunostaining was also observed in scleral blood vessels with the EP1 receptor antibody (not shown). A serial section from the same eye was labeled with secondary antibody alone (control) and showed no positive staining (Fig. 1C) . Using a nonserial section from the same donor eye, we detected positive FP receptor immunoreactivity in the scleral fibroblasts (Fig. 1L) and in scleral blood vessels. The pattern of labeling was similar to that observed with the EP1 receptor antibody. This experiment was repeated in a second donor eye with identical results (data not shown). In all sections examined, we did not detect any immunoreactivity above basal levels using the antibodies to the human EP2, EP3, or EP4 receptors (Figs. 1F 1H 1J , respectively). 
Positive immunoreactivity was observed in other tissues of the anterior segment. Specifically, the ciliary muscle contained immunoreactivity for the EP1, EP3, EP4, and FP receptors. In contrast, the ciliary epithelial cells contained EP1, EP2, and EP4 receptor immunoreactivity. Both the corneal epithelium and iris muscle had immunoreactivity to the EP1 and FP receptors; however, the intensity of this staining was strong in the corneal epithelium and weak in the iris muscle. These observations are in agreement with previous reports describing the localization of PG receptors in the anterior segment of the eye. 11 12 13 14  
Human PG Receptor Subtypes in Primary Cultured Scleral Fibroblasts
To evaluate the expression of PG receptors in HSFs, primary HSF cultures were established from tissue explants and immunostained using each of the prostanoid receptor antibodies. Immunoreactivity for the EP1, EP2, and FP receptors was detected in second-passage cultures of HSFs (Fig. 2) . Immunoreactivity for the EP1 and FP receptor antibodies was observed within every cell in the culture and was evenly distributed over the entire cell surface. In contrast, the immunoreactivity for the EP2 receptor antibody was much weaker and more diffuse and appeared to be primarily localized to the juxtanuclear region of the HSFs. This difference may account for the absence of EP2 immunoreactivity in the scleral sections. There was no difference in this staining pattern among second-, third-, or fourth-passage cultures. As with the tissue labeling, we were unable to detect EP3 and EP4 receptor immunoreactivity (Figs. 2D 2E) . These data are consistent with the labeling observed in tissue sections and further suggest that the receptor expression can be maintained in culture for several weeks. 
Expression of PG Receptor mRNA in Human Sclera
To evaluate transcription of PG receptor genes in sclera, RNA was extracted from human sclera, and the presence of messenger RNA encoding the EP1, EP2, and FP receptors was determined. RT-PCR was performed using primer sets specific for the EP1, EP2, and FP receptors that were predicted to yield the following PCR product sizes: 322 bp for EP1, 607 bp for EP2, and 1186 bp for the FP receptor. Figure 3 shows the PCR products obtained from these amplifications using total RNA isolated from sclera. In each case, lanes 1 and 2 of each panel show positive controls generated using human plasmid DNA encoding each of the PG receptors. Lanes 3 and 4 show the PCR products generated using RNA from human sclera. With PCR analysis, there were no mRNA transcripts for the EP3 and EP4 receptor subtypes in any of the scleral samples (data not shown). To confirm the identity of the PCR products, each product was incubated with restriction endonucleases to see whether predicted mobility shifts occur. Restriction digest of the EP1 receptor PCR product with AluI yielded the expected 192- and 85-bp fragments. Restriction digestion of the EP2 receptor PCR product with PstI yielded the expected 425-and 229-bp fragments. Restriction digest of the FP receptor PCR product with BamHI yielded the expected 825- and 361-bp fragments. Negative control experiments, containing all PCR reaction reagents except template, were performed for each combination of primers and did not yield any products (data not shown). In addition, because each primer pair was designed to span an intron, the PCR products did not result from the amplification of genomic DNA and thus are consistent with the presence of mRNA encoding the EP1, EP2, and FP receptor subtypes. 
Discussion
These studies demonstrate the localization and expression of EP1, EP2, and FP receptors in HSFs. Although the effects of PGs on the uveoscleral outflow pathway have not been characterized fully, they have been attributed to the activation of specific receptor subtypes present within uveoscleral outflow pathway. 15 16 In human eyes, Matsuo and Cynader 17 demonstrated specific binding sites for both PGF and PGE2 in the human ciliary muscle, but did not observe scleral binding sites. Ocklind et al. 18 found immunohistochemical FP receptor expression in the ciliary muscle and sclera of the monkey eye. 18 However, in situ hybridization experiments in the same study did not detect FP receptor transcripts in sclera. EP1, EP2, and EP4 receptors also have been identified in human ciliary muscle cells, using RT-PCR, by Mukhopadhyay et al. 13 The same investigators recently used in situ hybridization to identify mRNA transcripts for EP1 and FP receptors in the blood vessels of iris, the choroid, and ciliary muscle fibers in a human eye. 11 However, in neither of these studies were PG receptors observed in sclera. To the best of our knowledge, there has not been any prior observation of FP, EP1, and EP2 receptors with human sclera. Perhaps these prior methods did not have sufficient sensitivity to detect weak signals from the low-density scleral fibroblasts. 
PGF and latanoprost, a PGF analogue, reduce IOP by initiating a cascade of cellular events that lead to increased uveoscleral outflow. 19 20 21 22 Although the effects of this cascade are not fully understood, they may in part increase scleral permeability. Kim et al. 23 found that PG treatment of isolated sclera result in a dose-dependent and time-dependent increase in dextran permeability. This increase in transscleral permeability may be related to the decreased collagen immunoreactivity and increased matrix metalloproteinase (MMP) immunoreactivity that also have been observed in the sclera of monkey eyes after topical PGF-isopropyl ester treatment. 24 25 It is possible that one or more of the PG receptors on scleral fibroblasts mediate these changes. 
In conclusion, the data presented in this study demonstrate that EP1, EP2, and FP receptors are present in human scleral fibroblasts. It is not yet known whether activation of the EP1, EP2, or FP receptors influences MMP production by scleral fibroblasts or alter transscleral permeability. 
 
Table 1.
 
Prostaglandin Receptor Subtype-Specific Primers Used in the RT-PCR Reactions
Table 1.
 
Prostaglandin Receptor Subtype-Specific Primers Used in the RT-PCR Reactions
Primer GenBank Accession No. Nucleotide Sequence (bp) Codon Sequence (5′–3′)
hFP sense L24470 170–193 ATT TAG ACA GAA GTC CAA GGC ATC G
hFP antisense 1028–1057 GCA ACT GGT GAC TCA GAA ATA GCA GCA AAC
hEP1 sense L22647 969–991 CCT GTC GGT ATC ATG GTG GTG TC
hEP1 antisense 1269–1291 GGT TGT GCT TAG AAG TGG CTG AGG
hEP2 sense U119487 520–543 GCC ACG ATG CTC ATC CTC TTC GCC
hEP2 antisense 1151–1174 CTT GTG TTC TTA ATG AAA TCC GAC
hEP3 sense NM000957 665–692 GCA TAA CTG GGG CAA ACC TTT TCT TCG CC
hEP3 antisense 1022–1048 CTT AAC AGC AGG TAA ACC CAA GGA TCC
hEP4 sense D28472 520–543 GCC ACG ATG CTC ATC CTC TTC GCC
hEP4 antisense 1151–1174 CTT GTG TTC TTA ATC AAA TCC GAC
Figure 1.
 
Immunofluorescent labeling of EP1 and FP prostanoid receptor subtypes in human sclera. Serial sections (10 μm) of human sclera were fixed and labeled with primary antibodies against the EP1 (C, D), EP2 (E, F), EP3 (G, H), EP4 (I, J), and FP (K, L) receptors. Control sections (A, B) were labeled with secondary antibody alone. After incubation with rhodamine-conjugated secondary antibodies the sections were examined by epifluorescence microscopy, using a rhodamine band-pass filter (emission, 565 nm).
Figure 1.
 
Immunofluorescent labeling of EP1 and FP prostanoid receptor subtypes in human sclera. Serial sections (10 μm) of human sclera were fixed and labeled with primary antibodies against the EP1 (C, D), EP2 (E, F), EP3 (G, H), EP4 (I, J), and FP (K, L) receptors. Control sections (A, B) were labeled with secondary antibody alone. After incubation with rhodamine-conjugated secondary antibodies the sections were examined by epifluorescence microscopy, using a rhodamine band-pass filter (emission, 565 nm).
Figure 2.
 
Immunfluorescent labeling of prostanoid receptor subtypes in primary cultures of HSFs. Cells were cultured, fixed, and labeled with primary antibodies against the EP1 (B), EP2 (C), EP3 (D), EP4 (E), and FP (F) receptors. (A) Control culture of HSFs labeled with secondary antibody alone.
Figure 2.
 
Immunfluorescent labeling of prostanoid receptor subtypes in primary cultures of HSFs. Cells were cultured, fixed, and labeled with primary antibodies against the EP1 (B), EP2 (C), EP3 (D), EP4 (E), and FP (F) receptors. (A) Control culture of HSFs labeled with secondary antibody alone.
Figure 3.
 
RT-PCR with total RNA isolated from human sclera and amplified with specific primer sets for the human EP1, EP2, and FP receptors. Lane 1: products obtained after PCR using human plasmid DNA encoding the prostanoid EP1, EP2, and FP receptor subtypes; lane 3: products obtained after RT-PCR using total RNA isolated from human sclera; lanes 2, 4: fragments generated after restriction enzyme digestions with AluI for EP1, PstI for EP2, and BamHI for FP. Lanes S1, S2: Standards are 1-kb and 100-bp ladders (Gibco BRL). The predicted product sizes of the PCR products are as follows: EP1, 322 bp; EP2, 607 bp; and FP, 1180 bp.
Figure 3.
 
RT-PCR with total RNA isolated from human sclera and amplified with specific primer sets for the human EP1, EP2, and FP receptors. Lane 1: products obtained after PCR using human plasmid DNA encoding the prostanoid EP1, EP2, and FP receptor subtypes; lane 3: products obtained after RT-PCR using total RNA isolated from human sclera; lanes 2, 4: fragments generated after restriction enzyme digestions with AluI for EP1, PstI for EP2, and BamHI for FP. Lanes S1, S2: Standards are 1-kb and 100-bp ladders (Gibco BRL). The predicted product sizes of the PCR products are as follows: EP1, 322 bp; EP2, 607 bp; and FP, 1180 bp.
A portion of this work was prepared in partial fulfillment of the requirements for membership for Robert N. Weinreb in the American Ophthalmological Society. 
Coleman RA, Smith WL, Narumiya S. Classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev. 1994;46:205–229. [PubMed]
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Mukhopadhyay P, Bian L, Yin H, Bhattacherjee P, Paterson CA. Localization of EP1 and FP receptors in human ocular tissues by in situ hybridization. Invest Ophthalmol Vis Sci. 2001;42:424–428. [PubMed]
Ocklind A. Effect of latanoprost on the extracellular matrix of the ciliary muscle: a study on cultured cells and tissue sections. Exp Eye Res. 1998;67:179–191. [CrossRef] [PubMed]
Mukhopadhyay P, Geoghegan TE, Patil RV, Bhattacherjee P, Paterson CA. Detection of EP2, EP 4, and FP receptors in human ciliary epithelial and ciliary muscle cells. Biochem Pharmacol. 1997;53:1249–1255. [CrossRef] [PubMed]
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Stjernschantz J, Selen G, Sjoquist B, Resul B. Preclinical pharmacology of latanoprost, a phenyl-substituted PGF analogue. Adv Prostaglandin Thromboxane Leukot Res. 1995;23:513–518. [PubMed]
Woodward DF, Chan MF, Burke JA, et al. Studies on the ocular hypotensive effects of prostaglandin F ester prodrugs and receptor selective prostaglandin analogs. J Ocul Pharmacol. 1994;10:177–193. [CrossRef] [PubMed]
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Lindsey JD, Kashiwagi K, Boyle D, Kashiwagi F, Firestein GS, Weinreb RN. Prostaglandins increase proMMP-1 and proMMP-3 secretion by human ciliary smooth muscle cells. Curr Eye Res. 1996;15:869–875. [CrossRef] [PubMed]
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Figure 1.
 
Immunofluorescent labeling of EP1 and FP prostanoid receptor subtypes in human sclera. Serial sections (10 μm) of human sclera were fixed and labeled with primary antibodies against the EP1 (C, D), EP2 (E, F), EP3 (G, H), EP4 (I, J), and FP (K, L) receptors. Control sections (A, B) were labeled with secondary antibody alone. After incubation with rhodamine-conjugated secondary antibodies the sections were examined by epifluorescence microscopy, using a rhodamine band-pass filter (emission, 565 nm).
Figure 1.
 
Immunofluorescent labeling of EP1 and FP prostanoid receptor subtypes in human sclera. Serial sections (10 μm) of human sclera were fixed and labeled with primary antibodies against the EP1 (C, D), EP2 (E, F), EP3 (G, H), EP4 (I, J), and FP (K, L) receptors. Control sections (A, B) were labeled with secondary antibody alone. After incubation with rhodamine-conjugated secondary antibodies the sections were examined by epifluorescence microscopy, using a rhodamine band-pass filter (emission, 565 nm).
Figure 2.
 
Immunfluorescent labeling of prostanoid receptor subtypes in primary cultures of HSFs. Cells were cultured, fixed, and labeled with primary antibodies against the EP1 (B), EP2 (C), EP3 (D), EP4 (E), and FP (F) receptors. (A) Control culture of HSFs labeled with secondary antibody alone.
Figure 2.
 
Immunfluorescent labeling of prostanoid receptor subtypes in primary cultures of HSFs. Cells were cultured, fixed, and labeled with primary antibodies against the EP1 (B), EP2 (C), EP3 (D), EP4 (E), and FP (F) receptors. (A) Control culture of HSFs labeled with secondary antibody alone.
Figure 3.
 
RT-PCR with total RNA isolated from human sclera and amplified with specific primer sets for the human EP1, EP2, and FP receptors. Lane 1: products obtained after PCR using human plasmid DNA encoding the prostanoid EP1, EP2, and FP receptor subtypes; lane 3: products obtained after RT-PCR using total RNA isolated from human sclera; lanes 2, 4: fragments generated after restriction enzyme digestions with AluI for EP1, PstI for EP2, and BamHI for FP. Lanes S1, S2: Standards are 1-kb and 100-bp ladders (Gibco BRL). The predicted product sizes of the PCR products are as follows: EP1, 322 bp; EP2, 607 bp; and FP, 1180 bp.
Figure 3.
 
RT-PCR with total RNA isolated from human sclera and amplified with specific primer sets for the human EP1, EP2, and FP receptors. Lane 1: products obtained after PCR using human plasmid DNA encoding the prostanoid EP1, EP2, and FP receptor subtypes; lane 3: products obtained after RT-PCR using total RNA isolated from human sclera; lanes 2, 4: fragments generated after restriction enzyme digestions with AluI for EP1, PstI for EP2, and BamHI for FP. Lanes S1, S2: Standards are 1-kb and 100-bp ladders (Gibco BRL). The predicted product sizes of the PCR products are as follows: EP1, 322 bp; EP2, 607 bp; and FP, 1180 bp.
Table 1.
 
Prostaglandin Receptor Subtype-Specific Primers Used in the RT-PCR Reactions
Table 1.
 
Prostaglandin Receptor Subtype-Specific Primers Used in the RT-PCR Reactions
Primer GenBank Accession No. Nucleotide Sequence (bp) Codon Sequence (5′–3′)
hFP sense L24470 170–193 ATT TAG ACA GAA GTC CAA GGC ATC G
hFP antisense 1028–1057 GCA ACT GGT GAC TCA GAA ATA GCA GCA AAC
hEP1 sense L22647 969–991 CCT GTC GGT ATC ATG GTG GTG TC
hEP1 antisense 1269–1291 GGT TGT GCT TAG AAG TGG CTG AGG
hEP2 sense U119487 520–543 GCC ACG ATG CTC ATC CTC TTC GCC
hEP2 antisense 1151–1174 CTT GTG TTC TTA ATG AAA TCC GAC
hEP3 sense NM000957 665–692 GCA TAA CTG GGG CAA ACC TTT TCT TCG CC
hEP3 antisense 1022–1048 CTT AAC AGC AGG TAA ACC CAA GGA TCC
hEP4 sense D28472 520–543 GCC ACG ATG CTC ATC CTC TTC GCC
hEP4 antisense 1151–1174 CTT GTG TTC TTA ATC AAA TCC GAC
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