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1 From the Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany; and the 2 Department of Pediatrics, Philipps University, Marburg, Germany.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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METHODS. Expression of EP and FP receptor proteins was examined by immunohistochemistry on the light microscopic level, using subtype-specific antibodies on frozen and paraffin-embedded tissue sections of 10 normal human donor eyes. The subcellular distribution of the receptor proteins was studied by electron microscopic immunogold labeling. mRNA expression in various ocular tissues was analyzed by reverse transcription-polymerase chain reaction, using subtype-specific primers.
RESULTS. The highest expression of the EP1 receptor protein was found in the epithelia of the cornea, conjunctiva, lens, and the ciliary body; trabecular cells; iris vessels; and retinal ganglion cells. EP2 receptor labeling was most prominent in the corneal epithelium and choriocapillaries. EP3 and EP4 receptor labeling was primarily observed in the corneal endothelium and keratocytes, trabecular cells, ciliary epithelium, and conjunctival and iridal stroma cells, and EP3 was found, in addition, in retinal Müller cells. The highest expression of FP receptor protein was found in the corneal epithelium, ciliary epithelium, the circular portion of ciliary muscle, and iris stromal and smooth muscle cells. Immunoelectron microscopy showed a subcellular distribution of all prostanoid receptors along plasma membranes and the nuclear envelope. EP and FP receptor mRNA expression largely paralleled the proteins’ expression patterns.
CONCLUSIONS. The findings demonstrate a wide distribution but differential expression of FP and EP prostanoid receptor subtypes in human ocular tissues. EP1 through EP4 receptor subtype expression in human outflow pathways could be significant for future pharmacologic management strategies for the glaucomas.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In a previous study, we have demonstrated PGE2 to be a major PG in the aqueous humor of patients undergoing cataract surgery.6 The diverse actions of PGE2 are mediated by specific E-prostanoid (EP) receptors, which can be subdivided into at least four subtypes: EP1 through EP4.7 The four EP receptor subtypes differ in structure, binding profiles, and coupling to signal-transduction pathways.
EP receptors have been shown to be present in ocular cells and tissues of different animal species, mostly in the ciliary muscle or ciliary epithelial cells.8 9 10 11 12 13 14 15 Only a few studies have examined EP receptor localization in human ocular cell lines and isolated ocular cell types and tissues, such as lens epithelial cells16 or ciliary epithelial and ciliary muscle cells,17 18 19 20 21 22 by using molecular biological, radioligand-binding, or pharmacologic approaches. However, the specific distribution and precise localization of all four EP receptor subtypes in the human eye are not well characterized and have not yet been completely documented.
Because knowledge of the specific EP receptor distribution would provide a better understanding of the ocular effects of PGs and their analogues, we investigated the expression and precise cellular and subcellular localization in human ocular tissues of the four known EP receptors. For comparison, the PGF2
receptor FP was also studied. We used reverse transcription-polymerase chain reaction (RT-PCR) to identify specific EP receptor subtype mRNAs and immunohistochemistry with EP subtype-specific antibodies to localize the protein distribution on the light and electron microscopic level. This study is the first to demonstrate the presence and differential expression of all four EP prostanoid receptor subtypes and the FP receptor in all human ocular tissues and provides evidence for the existence of intracellular PG receptors, suggesting a role in gene transcription.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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For RT-PCR, fresh cornea, trabecular meshwork, iris, ciliary body including ciliary muscle, choroid, and retina from three normal-appearing donor eyes (ages 64, 71, and 81 years; two males, one female; 4 to 6 hours after death) and one eye surgically enucleated because of malignant melanoma of the posterior choroid (age 60 years; female) were dissected from surrounding tissues and homogenized. The protocol of the study adhered to the tenets of the Declaration of Helsinki for experiments involving human tissue.
Antibodies
Affinity-purified rabbit polyclonal antibodies raised against synthetic peptides specific for each EP receptor subtype were used in this study. Generation and characterization of the antibodies were performed as previously described.23
In addition, an affinity-purified rabbit polyclonal antibody raised against a synthetic peptide sequence from the human FP receptor (FP2: VYASDKEWIRFDQSNV) was used. Specificity of the antibody was determined by Western blot analysis and by preadsorption experiments.
Immunohistochemistry
The polyclonal antibodies (diluted 1:20–1:500) were used for immunolocalization of EP1, EP2, EP3, EP4, and FP receptor proteins on frozen sections, paraffin-embedded sections, and resin-embedded (LR White; Electron Microscopy Sciences, Fort Washington, PA) ultrathin sections of human ocular tissues, by means of immunofluorescence, immunoenzyme labeling, and postembedding immunogold labeling.
For indirect immunofluorescence, ocular tissues were embedded in optimal cutting temperature (OCT) compound and frozen in isopentane-cooled liquid nitrogen. Cryostat-cut sections (6 µm) were fixed in cold acetone, blocked with 10% normal goat serum, and incubated in primary antibody diluted in phosphate-buffered saline (PBS) overnight at 4°C. Antibody binding was detected by phalloidin-conjugated goat anti-rabbit secondary antibodies (Alexa 488; Molecular Probes, Eugene, OR).
For immunostaining of 4-µm paraffin-embedded sections, tissues were fixed in 4% paraformaldehyde in PBS at 4°C overnight and embedded in paraffin. Immunolabeling experiments were performed by the peroxidase-labeled streptavidin-biotin method (LSAB Plus-kit; DAKO, Glostrup, Denmark) according to the manufacturer’s instructions. 3-Amino 9-ethyl carbazol (AEC) was used as a chromogenic substrate and Mayer hemalun as a counterstain.
For postembedding immunogold labeling, tissue specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 to 5 hours at 4°C. Specimens were dehydrated serially to 70% ethanol at -20°C and embedded in resin (LR White; Electron Microscopy Sciences). Ultrathin sections were successively incubated in Tris-buffered saline (TBS), 0.05 M glycine in TBS, 0.5% ovalbumin and 0.5% fish gelatin in TBS, primary antibody diluted in TBS-ovalbumin overnight at 4°C, and finally 10 nm gold-conjugated secondary antibody (BioCell, Cardiff, Wales, UK) diluted 1:30 in TBS-ovalbumin for 1 hour. After they were rinsed, the sections were stained with uranyl acetate and examined with an electron microscope.
In negative control samples, the primary antibody was replaced by PBS or equimolar concentrations of nonimmune rabbit IgG or an irrelevant primary antibody.
Reverse Transcription-Polymerase Chain Reaction
Extraction of total RNA was performed with a kit (RNeasy Mini-Kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. After DNase I digestion, 1 µg total RNA was reverse transcribed in 20-µL reaction volumes, using 500 ng oligo dT primer and 200 U reverse transcriptase (SuperScript II; Life Technologies, Gaithersburg, MD).
Normalization of cDNAs from the different ocular tissues was performed in 20-µL PCR reaction volumes using primers for the housekeeping gene GAPDH and 2.5 µL of dilutions (1:5–1:50) of the first-strand products. Dilutions resulting in the same band intensity were used for analytic amplifications.
Specific intron-spanning primers were designed to amplify unique regions in the human EP receptor subtypes and the FP receptor (Table 1) . Amplification of EP and FP cDNAs was performed at the exponential phase in 25-µL reaction volumes, using the normalized template data and a program with a denaturation step of 95°C for 1 minute, and 40 cycles of 95°C for 15 seconds, 55°C (or 58°C and 61°C, respectively) for 30 seconds, and 72°C for 90 seconds. PCR products (10 µL) were analyzed in 1.2% agarose gels containing 250 ng/mL ethidium bromide. Images were obtained with an image-capture system (Cs-1; Cybertech, Berlin, Germany), and differences of band intensities were compared. Identity of the PCR fragments was subsequently confirmed by sequence analysis (Prism 310 gene sequence analyzer; PE-Applied Biosystems, Foster City, CA).
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Results |
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EP1 Receptor.
The antibody to the EP1 receptor subtype reacted strongly with all ocular tissues and predominantly labeled the epithelia of the cornea (Figs. 1A
6A)
, conjunctiva (Fig. 6B)
, lens (Fig. 6C) , and ciliary body (Figs. 4A
6E
6F)
, trabecular endothelial cells (Figs. 2A
6D)
, vascular endothelial cells of the iris (Figs. 3A
3B)
and retinal ganglion cells and photoreceptor cells (Figs. 5A
5B
6G
; Table 2
). In the trabecular meshwork, the trabecular cells, particularly in the outer portions of the meshwork, and the cells lining the Schlemm canal, collector channels, and scleral aqueous veins were prominently labeled (Figs. 2A
6D)
. Positive EP1 immunoreactivity in the iris was localized to vascular endothelial cells, and, to a lesser extent, to pigment epithelial cells, dilator and sphincter muscle cells, and individual stromal cells, presumably melanocytes (Fig. 3A)
. In the ciliary body, nonpigmented and pigmented epithelial layers showed an equally prominent immunoreaction, particularly at the ciliary crests (Figs. 4A
6E)
, whereas the ciliary muscle showed moderate labeling intensity (Fig. 6F)
. In the neuroretina, all layers were weakly positive, with the most prominent staining in the ganglion cell layer, the inner nuclear layer, and the photoreceptor inner segments, whereas the photoreceptor outer segments and Müller cells were completely negative (Figs. 5A 5B
6G)
. Positive immunoreactivity was also detected in the endothelial cells of the retinal vasculature.
EP2 Receptor.
EP2 receptor labeling was only moderately present in ocular tissues. The most prominent staining for the EP2 receptor protein was observed in the corneal epithelium (Fig. 1B)
; the conjunctival epithelium at the limbus, notably in its basal layers (Fig. 1F) ; and the vascular endothelium of the choriocapillaries (Fig. 5C
; Table 2
). Whereas trabecular cells were only weakly labeled, the outer wall of the Schlemm canal and scleral cells in the immediate periphery of the outer wall, collector channels, and scleral aqueous veins showed more intense immunoreactions (Fig. 2B)
. Specific EP2 labeling was also observed on the ciliary epithelium, which showed a dotlike labeling pattern along the basal aspects of the pigmented layer in the pars plicata and of both pigmented and nonpigmented layers in the pars plana of the ciliary body (not shown). The ciliary muscle stained only weakly. In the neuroretina, staining appeared weak and diffuse throughout all layers, but was most abundant in the ganglion cell and nerve fiber layers as well as retinal capillaries (Fig. 5C)
. In addition to individual stromal cells, immunopositive fiberlike structures resembling nerve fibers or cell processes were generally observed in the sclera, the trabecular meshwork, and the iris stroma.
EP3 Receptor.
EP3 receptor subtype labeling was primarily observed in the corneal endothelium and keratocytes (Fig. 1C)
; in endothelial cells of the trabecular meshwork (Figs. 2C
2D)
; in the ciliary epithelium (Fig. 4B)
; in stromal cells of the conjunctiva, particularly in the limbal area (Figs. 1G
1H)
; in the iris (Figs. 3C
3D)
; and in Müller cells of the retina (Fig. 5D) . The trabecular endothelial cells showed a marked immunopositive reaction, whereas endothelial cells lining the Schlemm canal and collector channels were moderately positive (Figs. 2C
2D)
. Scleral cells in the periphery of collector channels and aqueous veins were also labeled. The iris showed a moderate reaction of dilator and sphincter muscles, but a prominent perinuclear labeling pattern of stromal cells (Figs. 3C
3D)
. Part of the vessels in the iris stroma, particularly in the iris root, were also positive. In the ciliary body, most of the immunoreaction was located in the basal aspects of the pigmented epithelial cells in the pars plicata (Fig. 4B)
and in the nonpigmented epithelial cells in the pars plana of the ciliary body. Individual muscle cells in the ciliary body were distinctly positive. Strong and specific labeling was exclusively present on Müller cells and their processes within the retinal layers, whereas all neuronal structures appeared to be largely negative and retinal vessels only weakly positive (Fig. 5D)
. In addition to cellular staining, immunopositive fiberlike structures resembling nerve fibers or cell processes were observed in the sclera; in the conjunctival stroma penetrating the basal layer of the conjunctival epithelium (Fig. 1G)
; in the stroma of iris (Fig. 3C) , ciliary body, and choroid; and in the ciliary muscle (Fig. 4E) .
EP4 Receptor.
The strongest expression of the EP4 receptor protein was observed in the corneal endothelium and keratocytes (Fig. 1D)
, in endothelial cells of trabecular meshwork (Fig. 2E)
, in the ciliary epithelium (Fig. 4C
4D)
, and in cells of the iris stroma (Fig. 3E)
and conjunctival stroma in the limbal region. In the trabecular meshwork, prominent immunoreactivity was present on the trabecular cells and endothelial cells lining the Schlemm canal, collector channels, and aqueous veins (Fig. 2E)
. In addition to smooth muscle cells and stromal cells of the iris (Fig. 3E)
, part of the iridal vessels were markedly positive, particularly in the iris root area. In the ciliary body, the basal aspects of the pigmented epithelium were found to be strongly labeled in the pars plicata (Fig. 4C)
, whereas the nonpigmented epithelium was markedly positive in the pars plana region (Fig. 4D)
. The ciliary muscle showed only moderate immunopositivity, but individual muscle cells and blood vessels within and anterior to the ciliary muscle were heavily labeled. Blood vessels in the ciliary processes were negative, however. In the retina, prominent labeling was limited to the nerve fiber layer, whereas all other retinal layers were only weakly labeled (Fig. 5E)
. Müller cells and retinal vessels were essentially negative. Strongly immunofluorescent globular structures resembling corpora amylacea were found occasionally in the inner plexiform layer. Fiberlike immunopositive structures resembling nerve fibers were again observed in the stroma of iris (Fig. 3E)
, ciliary body, and choroid; in the ciliary muscle; and in the sclera surrounding blood vessels.
FP Receptor.
The highest expression of FP receptor protein was found in the corneal epithelium, revealing a clear perinuclear labeling pattern (Fig. 1E)
; in the ciliary epithelium; in the circular portion of the ciliary muscle (Fig. 4F)
; and in the stromal cells and smooth muscle cells of the iris, particularly the sphincter muscle (Fig. 3F)
. The most striking feature of FP receptor-like immunoreactivity was the focal labeling of individual cells (e.g., in the conjunctival stroma at the limbal region, the corneal stroma, the sclera, the iris stroma, the ciliary muscle, and the trabecular meshwork).
Trabecular meshwork cells, particularly in the outer portions of the meshwork and endothelial cells of the Schlemm canal, collector channels, and aqueous veins showed moderate immunopositivity (Fig. 2F) . Positive immunoreactivity was also localized to smooth muscle cells and individual cells in the iris stroma, presumably melanocytes (Fig. 3F) , particularly concentrated along the anterior border layer. Specific labeling was also detected in both pigmented and nonpigmented layers of the ciliary epithelium, particularly in the pars plicata. In addition, specific immunoreactivity was detected in some stromal cells anterior to the ciliary muscle, in individual ciliary muscle cells (Fig. 4F) , and in vascular endothelial cells lining blood vessels within and anterior to the ciliary muscle, whereas capillaries in the ciliary processes were negative. The choroidal stroma had groups of large, heavily stained cells resembling ganglion cells adjacent to large vessels in the deeper stroma (Fig. 5F) . In the retina, all neuroretinal layers and retinal vessels were weakly positive, whereas Müller cells appeared to be unstained. Brightly fluorescent globular structures resembling corpora amylacea were present in the ganglion cell layer and inner plexiform layer.
Electron Microscopy.
Immunogold labeling confirmed positive cellular staining for EP and FP receptor proteins in all ocular cell types (e.g., in corneal epithelial cells; Fig. 7A
), trabecular endothelial cells and the endothelia of the Schlemm canal (Fig. 7B)
, iris stromal melanocytes (Fig. 7C)
, vascular endothelial cells (Fig. 7D) , and ciliary nonpigmented (Figs. 7E
7F)
and pigmented epithelial cells (Fig. 7G)
.
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Expression of EP and FP Receptor mRNA in Human Ocular Tissues
Reverse transcription followed by PCR was performed, using total RNA extracted from different ocular tissues and primer sets specific for the individual FP and EP receptor subtypes. The PCR reactions with the RNA isolated from the three donor eyes and the eye surgically enucleated for melanoma yielded largely identical results. Fig. 8
shows ethidium bromide-stained agarose gels with the PCR products obtained with total RNA isolated from two representative human eyes. The identity of the PCR products of predicted size was confirmed by sequence analysis. Negative control experiments without templates did not yield any products (not shown).
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When the tissue-specific mRNA expression of the different receptor subtypes for each eye were compared, the cornea showed uniformly the highest expression of EP3, followed by EP4 and FP, and the lowest expression of EP1 and EP2. This largely paralleled the protein expression, which was most prominent for EP3 and EP4, and weaker for EP1, EP2, and FP. mRNA expression in the trabecular meshwork differed somewhat between the eyes: Whereas the highest expression was consistently found for the EP1 and EP3 subtypes, expression of EP2, EP4, and FP showed interindividual differences with low expression of EP2 and EP4 in two of the eyes, and low expression of EP4 and FP in the other two. The mRNA expression in the iris showed the highest levels for EP3, EP4, and FP and lower expression levels for EP1 and EP2, again reflecting the general expression pattern of the proteins. The ciliary body showed consistently in all four eyes the highest mRNA expression for the FP receptor, followed by an equal level of expression of EP1, EP3, and EP4, and the lowest expression for EP2. This expression pattern was well reflected by the general expression pattern of the proteins. mRNA expression in the retina of all four eyes was highest for the EP3 receptor, followed by EP1 and FP, and was lowest for EP2 and EP4, again matching the expression patterns of the proteins. The choroid showed a consistent expression pattern for mRNA in all eyes analyzed, with an equally high expression of mRNA for EP3, EP4, and FP, and a lower expression for EP2 and EP1, again matching the expression pattern of the proteins.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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in cornea, conjunctiva, sclera, trabecular meshwork, lens, iris, ciliary body, retina, and choroid of the human eye. Whereas RT-PCR identified receptor mRNA expression in the various tissues, immunolabeling with subtype-specific antibodies permitted a more precise localization of tissue distribution and subcellular localization of receptor proteins. The immunohistochemical findings were largely in agreement with the molecular biological findings, and marked differences between mRNA and protein distribution, as reported in a previous study,24
were not noted.
Distribution of PG Receptors in Ocular Tissues
Prior studies in animals have identified prostanoid EP and FP receptors in ocular tissues, primarily the iris-ciliary body complex, using radioligand binding and functional assays,8
9
10
11
12
13
14
15
24
25
but comparison of these results with the present findings in human tissues is difficult. For instance, all four EP receptor subtypes were shown to be expressed in bovine ciliary epithelium14
: The EP1 and EP4 receptors were found primarily in nonpigmented epithelial cells, whereas the EP2 receptor was localized to the pigmented epithelial cells, and the EP3 receptor subtype was localized to both the pigmented and the nonpigmented cells. This distribution differs from the human situation, in which all four EP receptor subtypes were obviously expressed in both epithelial layers, predominantly in the pigmented layer in the pars plicata and in the nonpigmented layer in the pars plana of the ciliary body. However, the findings of FP receptor distribution in monkey eyes largely corresponds to the present findings in human ocular tissues: FP receptor protein and message were found to be most prominent in the corneal and conjunctival epithelium and in the ciliary muscle and ciliary processes and to be moderately prominent in the retina, iris, and connective tissues of monkey eyes.24
Molecular biological and functional studies on prostanoid receptor distribution in human ocular tissues in situ and in vitro have shown that trabecular meshwork cells express FP receptors,26 lens epithelium expresses EP4 and FP receptors,16 and ciliary epithelial and ciliary muscle cells express EP2, EP4, and FP receptors.17 18 19 20 21 27 These isolated data are largely confirmed by the findings of the present study. However, it has to be taken into account that the literature is somewhat confusing regarding the EP2 receptor’s nomenclature, because before 1995, when the human EP2 receptor was cloned, the cloned EP4 receptor was misclassified as the EP2 receptor.28 Ligand binding and quantitative autoradiographic visualization of FP receptors yielded the highest receptor concentration in the longitudinal ciliary muscle, the iris sphincter muscle, and the retina of human eyes,29 which is partly in agreement with the present findings; however, in this study, high concentrations of FP receptors were mainly detected in the circular portion of the ciliary muscle and also in the corneal and ciliary epithelium. In situ hybridization revealed the presence of high levels of EP1 receptor mRNA in blood vessels of the iris, ciliary body, and choroid, in the iris sphincter, ciliary muscle, and retina (photoreceptors, inner and outer nuclear layers, ganglion cells). FP receptor mRNA was predominantly present in the circular portion of the ciliary muscle and in the ciliary stroma, iris vasculature, and iris sphincter, but was absent in the retina and choroidal vasculature.22 Apart from the absence of signals for FP receptor mRNA in retina and choroid, these data are again in good agreement with the findings of receptor distribution in the present study.
The present study also provides evidence for the existence of intracellular PG receptors EP1 through EP4 and FP in the nuclear envelope of various ocular cell types, suggesting an intracellular action of PGs and a direct influence of PGs on gene transcription. These findings are supported by recent data in the literature showing nuclear association of functional EP1, EP3, and EP4 receptors in porcine cerebral tissue (i.e., in vascular endothelial cells and neurons), in fibroblast cell lines, and in human embryonic kidney cells.30 31 Stimulation of these nuclear receptors has been shown to modulate nuclear calcium and gene transcription. Consistent with the recently demonstrated localization of cyclooxygenase-2 in the perinuclear region of ocular cells,6 it is conceivable that locally produced intracellular PGs can activate nuclear EP receptors and modulate gene transcription in various ocular cell types.
Functional Significance of PG Receptors in Ocular Tissues
Prostanoid FP and EP receptor subtypes are coupled to different signal transduction systems, and stimulation of these receptors triggers different cellular signals: FP and EP1 stimulate intracellular Ca2+ mobilization, EP3 inhibits adenylate cyclase, and EP2 and EP4 activate adenylate cyclase.3
Thus, FP, EP1, and EP3 receptors are generally considered constrictor receptors, whereas EP2 and EP4 are considered receptors with relaxant properties. In view of these specific signal-transduction mechanisms, knowledge of the specific PG receptor distribution is needed to facilitate interpretation of the action of substances.
PGE2, the major PG in human aqueous humor,6 has a broad range of biological actions, including the contraction or relaxation of vascular and nonvascular smooth muscles, stimulation or suppression of neurotransmitter secretion, regulation of intraocular pressure, and regulation of cell growth.7 PGE1 has been shown to stimulate growth and melanogenesis of iris melanocytes, by acting on the EP2 receptor.32 Cell-type-specific signal-transduction pathways may account for the diverse biological effects induced by PGE. The presence and differential distribution of various EP receptor subtypes in human ocular tissues, as demonstrated in this study, offer a potential explanation for the multiple effects of PGE2.
Topically administered PGs reduce intraocular pressure in animals and in normal and glaucomatous human eyes.1
2
4
5
It is generally accepted that PGF2 analogues such as latanoprost reduce intraocular pressure mainly by increasing the uveoscleral outflow,33
34
perhaps through the mediation of FP receptors on ciliary muscle cells.18
20
22
27
35
36
A high expression of FP receptor mRNA in the ciliary body and a prominent localization of FP receptor proteins in the circular portion of the human ciliary muscle, but only moderate expression and localization in the trabecular meshwork, were confirmed by the present study and are consistent with this proposed mechanism of action. The increase in uveoscleral outflow is thought to occur by activation of intracellular signal-transduction pathways, including cAMP formation, and induction of c-Fos and c-Jun expression. These signals are believed to cause increased synthesis of matrix metalloproteinases and, consistently, increased degradation of extracellular matrix components in the intercellular spaces.36
However, it is also conceivable that induction of the nuclear transcription factors c-Fos and c-Jun occurs by direct intracellular action of PGs on nuclear prostanoid receptors, influencing gene transcription and induction of metalloproteinase production.
Topically applied PGE2 and its analogues also markedly reduce intraocular pressure in humans and animal species, which may be preceded by a transient pressure elevation,2
37
38
but the underlying mechanism of the pressure-lowering effects of PGE2 is, in contrast to that of PGF2, not fully understood. Studies in the perfused human anterior segment have shown that PGE1 causes a dose-dependent increase in trabecular outflow,39
suggesting different mechanisms of action for PGF2 and PGE1. Intraocular pressure reduction in animals was shown to be aided by selective agonists for most prostanoid EP receptors, implying the involvement of EP1, EP2, and EP3 receptor subtypes,40
41
and the presence of EP2 receptors in the bovine trabecular meshwork and ciliary muscle has been suggested by a functional study.42
Specific prostanoid receptors present on human trabecular meshwork cells may mediate some of the pressure-lowering effect of PGE. Consistent with this, a high expression of EP1, EP2, EP3, and EP4 receptor mRNA in the human trabecular meshwork and expression of all EP receptor proteins, most prominently EP1, EP3, and EP4, in trabecular cells and endothelial cells lining the Schlemm canal and collector channels was demonstrated in this study. Whereas the expression of FP receptors on human and bovine trabecular cells has already been reported,26
42
the additional presence of all EP receptor subtypes on human trabecular cells may be important for future pharmacologic management strategies for the glaucomas. Based on the present findings, EP1, EP3, and EP4 receptor agonists, in particular, may substantially affect aqueous outflow and may be promising ocular hypotensive agents.
However, the ocular hypotensive action of PGE2 and its analogues may be due not only to an increased trabecular outflow facility, because all EP receptor subtypes are also expressed in the human ciliary muscle (although to a lesser extent than the FP receptor) and may thus also contribute to regulation of uveoscleral aqueous outflow. Stimulation of EP2 and EP4 receptors is thought to mediate relaxation of smooth muscle cells, whereas stimulation of EP1 and EP3 receptors is believed to cause smooth muscle contraction.3 7 Thus, the coexistence of all four EP receptor subtypes in human ciliary muscle and trabecular cells, which also possess smooth muscle-like contractile properties,43 suggests that regulation of aqueous outflow by PGE2 is based on complex antagonistic mechanisms involving multiple receptor subtypes. However, mechanisms of action other than contraction of smooth muscle cells are also possible.
There is also evidence that that a significant proportion of the ocular hypotensive action of PGF2 and its analogues is attributed to an additional PGE2 agonistic activity, mainly mediated by EP1 and EP3 receptors,3
41
both of which were shown to be prominently expressed in the human trabecular meshwork and ciliary muscle.
Although the intraocular-pressure-reducing effect of PGs and their analogues has been well documented, the microvascular effects are less well known.3
44
Wide expression of EP and FP receptors in vascular endothelial and smooth muscle cells in virtually all human ocular tissues examined suggests that EP and FP receptors also mediate vascular reactions of PGF2 and PGE2. Although the EP1 receptor has been detected immunohistochemically in blood vessels of the human conjunctiva, sclera, iris, ciliary body, choroid, and retina in the present study, functional studies in monkey eyes have suggested that neither the EP1 nor the FP receptor is involved in the regulation of ocular blood flow.3
However, a selective EP2 receptor agonist causes a marked drop in vascular resistance of the choroid, indicating that EP2 receptors mediate vasodilation in the primate eye. This assumption is supported by the prominent immunolabeling for the EP2 receptor subtype in choriocapillaries and retinal vessels in human eyes, as shown in the current study. Based on the present findings, the receptors thought to be involved in mediating the effects of PGE2 on the retinal and choroidal microvasculature of human eyes are predominantly of the EP1 and EP2 subtype that may elicit vasoconstriction and vasodilation, respectively. This observation is in agreement with findings in pig eyes: Although porcine retinal vessels have been described to contain EP1, EP2, EP3, and FP receptors, the receptors mainly involved in regulating retinal and choroidal blood flow are the EP1 and EP2 receptor subtypes.45
Although both EP2 and EP3 receptors have been detected on porcine neurites,46 only the EP2 subtype was found in plexiform and nerve fiber layers of the human retina, whereas the EP3 subtype appeared to be confined to Müller cells. However, the additional presence of EP3 receptors on neuroretinal structures cannot be completely excluded, because a clear differentiation between Müller cell processes and nerve fibers was not feasible on the light microscopic level. The significance of the expression of EP receptors in the cornea, conjunctiva, sclera, and lens, is not yet known.
In conclusion, all four known EP receptor and FP receptor proteins and mRNAs were found to be widely distributed but differentially expressed in human ocular tissues, suggesting an array of effects of PGE2 and PGF2 in the eye. Although the functional significance and precise roles of specific EP receptor subtypes in the human eye remain to be determined, the findings in the present study provide detailed baseline data for the assessment of the pathophysiological roles of these prostanoid receptors and could be an important tool to facilitate the use of EP subtype-specific agonists or antagonists in human ocular diseases.
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Acknowledgements |
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Footnotes |
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Submitted for publication August 10, 2001; revised January 10, 2002; accepted January 25, 2002.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Ursula Schlötzer-Schrehardt, Department of Ophthalmology, University of Erlangen-Nürnberg, Schwabachanlage 6, D-91054 Erlangen, Germany; ursula.schloetzer{at}augen.imed.uni-erlangen.de.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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in the human eye Graefes Arch Clin Exp Ophthalmol 222,139-141[Medline][Order article via Infotrieve]
Curr Eye Res 1,205-209[Medline][Order article via Infotrieve]
binding sites in the bovine iris ciliary body Invest Ophthalmol Vis Sci 34,2237-2245 receptor messenger RNA and protein in the cynomolgus monkey eye Invest Ophthalmol Vis Sci 37,716-726 receptors in the human trabecular meshwork Invest Ophthalmol Vis Sci 39,315-321 on calcium efflux Invest Ophthalmol Vis Sci 33,2679-2686 and latanoprost on cultured human iridal melanocytes Exp Eye Res 70,113-120[Medline][Order article via Infotrieve]
on aqueous humor dynamics in cynomolgus monkeys Curr Eye Res 6,1035-1044[Medline][Order article via Infotrieve]
Exp Eye Res 47,761-769[Medline][Order article via Infotrieve]
receptors on the retinal vasculature of the pig Prostaglandins 50,253-267[Medline][Order article via Infotrieve]
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