Effects of Prostanoid EP Agonists on Mouse Intraocular Pressure

Tadashiro Saeki; Takashi Ota; Makoto Aihara; Makoto Araie

doi:10.1167/iovs.08-2800

Abstract

purpose. To investigate the ocular hypotensive effect and change of outflow facilities by prostaglandin E2 (PGE2) and selective agonists of PGE2 receptor subtypes (EP1, -2, -3, -4) using C57BL/6 (wild-type [WT]) mice or FP, EP1, -2, and -3 receptor–deficient mice.

methods. IOP was measured with a microneedle, and IOP reduction was evaluated by the difference in IOP between the treated eye and the contralateral control eye. The time course and dose dependency of IOP reduction with PGE2 and the four selective EP receptor agonists were assessed. Aqueous humor outflow facility was measured by a two-level constant-pressure perfusion method.

results. PGE2, ONO-AE1–259-01 (EP2 agonist), and ONO-AE1-329 (EP4 agonist) significantly reduced IOP in a dose-dependent manner, whereas ONO-DI-004 (EP1 agonist) and ONO-AE-248 (EP3 agonist) had no effect. Peak IOP reduction at 2 hours with 0.1% ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) were 21.1% ± 4.8% and 17.5% ± 2.9%, respectively (P < 0.01). IOP reduction with ONO-AE1-259-01 (EP2 agonist) was completely eliminated in EP2 knockout mice, but IOP reduction in other knockout mice was similar to that observed in WT mice. The effects of ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) on the outflow facility were similar to those of their carrier.

conclusions. EP2 and EP4 receptors mediated IOP reduction in mice, whereas the contribution of EP1 and EP3 receptors was insignificant. The EP2 and EP4 receptor–mediated mechanisms of IOP reduction were different from those mediated by the FP receptor.

Eight kinds of prostanoid receptors are present as functional receptors for prostaglandins (PGs), which consist of varieties of lipid mediators related to systemic homeostasis or disorders.¹In ophthalmology, PGs have been identified as ocular inflammatory mediators.² ³ ⁴However, in the 1980s, PGs were found to reduce intraocular pressure (IOP). PGF2α isopropyl ester⁵and unoprostone, a metabolic product of PGF2α,⁶were first introduced for use in human eyes. Since then, four additional types of PG analogues have been developed, and there are now five types on the market for glaucoma treatment. Based on the observation that their IOP-lowering effects were completely abolished in FP receptor knockout mice, it was shown recently that the common and indispensable prostanoid receptor for these five PG analogues is FP.⁷ ⁸However, it has been suggested that other prostanoid receptors may also be involved in aqueous humor dynamics. Several studies have shown that PGE2, a nonspecific EP agonist, has a strong potential for IOP reduction.⁹ ¹⁰ ¹¹ ¹²Moreover, FP-induced endogenous PGE2 production was detected in the anterior segment,¹³ ¹⁴and PGE2 was reported to induce matrix metalloproteinases,¹⁵which may also increase the outflow of aqueous humor through the uveoscleral outflow pathway in response to PG analogues.¹⁶Four types of PGE2 receptors have been identified. They are EP1, -2, -3, and -4. Interestingly, the EP3 receptor subtype was shown to be involved in FP-induced IOP reduction through endogenously induced PGE2.¹⁷Thus, further clarification of the roles of EP receptors in aqueous humor dynamics is of special interest. Recently, specific agonists for each EP receptor subtype (EP1–EP4) and EP1, -2, and -3 knockout mice have become available. In this study, the ocular hypotensive effects of a specific agonist for each EP receptor subtype in addition to PGE2 were studied in FP, EP1, -2, and -3 receptor–deficient mice and wild-type (WT) mice. Furthermore, the effects of EP2 and EP4 agonists and latanoprost on the aqueous humor outflow facility were determined in WT mice.

Materials and Methods

Animals

All experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 (WT) mice, the background strain of the knockout mice used, were purchased from Japan SLC (Hamamatsu, Japan) at 5 weeks of age and used as the WT control. Mouse genes encoding the FP, EP1, -2, or -3 receptor were disrupted by gene knockout methods using homologous recombination, as reported previously,¹⁸ ¹⁹ ²⁰and FP, EP1, EP2, or EP3-homozygous knockout (FPKO, EP1KO, EP2KO, or EP3KO) mice were used. The EP4-homozygous knockout (EP4KO) mice could not be used because the mutation has a high mortality rate (more than 95% neonatal death within 72 hours of birth).²¹Mice were bred and housed in clear cages covered loosely with air filters and containing white chip bedding. The temperature was kept at 21°C with a 12-hour light (6:00 AM–6:00 PM) and 12-hour dark cycle. All mice were fed ad libitum and were acclimatized to the environment for at least 2 weeks before experimentation. In all experiments, we used mice older than 8 weeks of age.

Preparation and Instillation of Ophthalmic Solution

PGE2 methyl ester was purchased from Cayman Chemical Co. (Ann Arbor, MI) and was dissolved in 5% dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS). Selective agonists to each EP receptor subtype (ONO-DI-004, EP1 agonist; ONO-AE1-259-01, EP2 agonist; ONO-AE-248, EP3 agonist; ONO-AE1-329, EP4 agonist) were donated by Ono Pharmaceutical (Osaka, Japan), and latanoprost (0.005%) was purchased from the hospital pharmacy. The specificity of the respective EP agonists was analyzed by measuring their binding affinity to the respective EP receptors expressed in Chinese hamster ovary cells.²²The high specificity of each agonist for each EP receptor subtype was confirmed²³as follows: ONO-DI-004 is the EP1 receptor–selective compound, exhibiting a high affinity (inhibition constant, K_i = 150 nM) for the EP1 receptor, and low affinity (K_i > 3.3 × 10³ nM) for the other prostanoid receptors (EP2, EP3, EP4, DP, FP, IP, TP). ONO-AE-259-01, the EP2 receptor–selective compound, exhibits high affinity (K_i = 3 nM) for the EP2 receptor, moderate affinity (K_i = 140 nM) for the DP receptor, and low affinity (K_i > 3.3 × 10³ nM) for the other prostanoid receptors (EP1, EP3, EP4, FP, IP, TP). ONO-AE-248, the EP3 receptor–selective compound, exhibits high affinity (K_i = 7.5 nM) for the EP3 receptor and low affinity (K_i > 3.3 × 10³ nM) for the other prostanoid receptors (EP1, EP2, EP4, DP, FP, IP, TP). ONO-AE1-329, the EP4 receptor–selective compound, exhibits high affinity (K_i = 10 nM) for the EP4 receptor, moderate affinity (K_i = 2100 and 1200 nM) for the EP2 and EP3 receptor, respectively, and low affinity (K_i > 3.3 × 10³ nM) for the other prostanoid receptors (EP1, DP, FP, IP, TP). PGE2 has high affinity (K_i = 20, 12, 1, 2, and 2.4 nM) for the EP1, -2, -3, -4, and DP receptors, respectively, and moderate affinity (K_i = 100 nM) for the FP receptor. EP agonists were stored at −80°C in 100% DMSO and were diluted with PBS just before use to yield a 5% DMSO concentration. With a micropipette, 3 μL of each drug solution was topically applied in a masked manner to one eye selected at random, and the carrier solution (PBS with 5% DMSO) was applied to the other eye as a control.

IOP Measurement

IOP was measured with a microneedle in mice anesthetized with ketamine and xylazine, as described previously.¹⁷ ²⁴ ²⁵Briefly, a microneedle made of borosilicate glass (100-μm tip diameter, 1-mm outer diameter; World Precision Instruments Inc., Sarasota, FL) was connected to a pressure transducer (model BLPR; World Precision Instruments Inc.). The pressure measured by the transducer was recorded on a data acquisition and analysis system (PowerLab; ADInstruments, Colorado Springs, CO). The microneedle was placed in the anterior chamber, and the conducted pressure was recorded in both eyes during a window of 4 to 7 minutes after anesthesia. Given that a higher baseline IOP has been reported at night in each genotype,¹⁷all measurements were performed from 9:00 to 11:00 PM and under red light illumination to eliminate the effect of lighting on IOP. The effect of each drug was calculated as the ratio of IOP reduction, defined as 100 × (IOP of treated eye − IOP of contralateral carrier–treated eye)/IOP of contralateral carrier–treated eye (%) in each mouse and is termed % IOP change.

Effects of ONO-AE1-259-01 (EP2 Agonist), ONO-AE1-329 (EP4 Agonist), and Latanoprost on Outflow Facility

To clarify the mechanism of IOP reduction with EP agonists, the aqueous humor outflow facility in WT mice was measured at 2 hours after the administration of 0.1% ONO-AE1-259-01 (EP2 agonist), ONO-AE1-329 (EP4 agonist), or the carrier solution or at 3 hours after the topical administration of 0.005% latanoprost or its carrier solution. Latanoprost was used as a positive control to increase the outflow facility.²⁶Each time point was selected because the maximum IOP reduction with each agent was observed at that time.

Total outflow facility was measured by a two-level, constant-pressure perfusion method²⁶ ²⁷ ²⁸and was reported as the C value. Briefly, the infusion needle inserted into the anterior chamber was connected to the reservoir (0.35-mm inner diameter polyethylene tube; Natsume Seisakujo, Tokyo, Japan) filled with artificial aqueous humor (BSS Plus; Alcon, Fort Worth, TX) through the pressure transducer.

A change in the height of the surface of artificial aqueous humor after IOP was maintained at 25 or 35 mm Hg for 10 minutes at steady state (H₂₅ or H₃₅, respectively, in mm). The inner cross-sectional area of the reservoir (S, in square mm) was calculated from the inner diameter. The volume of artificial aqueous humor infused per minute at 25 or 35 mm Hg was defined as V₂₅ or V₃₅ (mL/min). The total outflow facility (C_total, μL/min/mm Hg) was calculated as follows²⁶ ²⁷ ²⁸ : C_total = (V₃₅ − V₂₅)/10 = [S(H₃₅ − H₂₅)]/100. The total outflow facility (C_total) calculated above by the two-level constant-pressure perfusion method was the sum of C_conv and C_uveo, when C_conv indicated outflow facility of the pressure-dependent outflow system and C_uveo indicated that of the uveoscleral outflow system, which was assumed to be less pressure dependent.²⁹ ³⁰

Statistical Analysis

The Mann-Whitney U test and the Wilcoxon signed rank test were used as appropriate. The Kruskal Wallis test and the Steel test were used for multiple comparisons as appropriate. P < 0.05 was considered statistically significant. All data were expressed as mean ± SEM.

Results

Time-Dependent IOP-Lowering Effects of PGE2 Methyl Ester in WT Mice

IOP was measured in WT mice at 1, 2, 3, or 6 hours after the application of 0.1% PGE2 methyl ester (n = 10 at each time point). IOP in the treated eyes was compared with that in the contralateral carrier–treated eyes by the Wilcoxon signed-rank test. IOP reductions with 0.1% PGE2 methyl ester at 1, 2, 3, or 6 hours after administration were 22.9% ± 4.5% (P = 0.0051), 25.5% ± 3.0% (P = 0.0051), 26.3% ± 2.0% (P = 0.0051), and 18.3% ± 1.5% (P = 0.0050), respectively. PGE2 increased IOP at 1 hour but decreased it after 2 hours. The application of 0.1% PGE2 methyl ester showed a maximum IOP reduction at 2 to 3 hours after administration (Fig. 1) .

Dose-Dependent IOP-Lowering Effect of PGE2 Methyl Ester in WT Mice

The dose-dependent effect of PGE2 methyl ester on IOP reduction was assessed with 0.01%, 0.001%, and 0.0001% concentrations at 3 hours after application, when 0.1% PGE2 methyl ester showed the maximum IOP reduction (n = 8 at each dose). IOP reductions with 0.1%, 0.01%, 0.001%, and 0.0001% PGE2 methyl ester compared with the carrier were 26.3% ± 2.0% (P = 0.0051), 15.7% ± 2.4% (P = 0.0117), 6.1% ± 3.4% (P = 0.1614), and 0.4% ± 2.3% (P > 0.90), respectively (Wilcoxon signed-rank test; Fig. 2 ). PGE2 methyl ester significantly lowered IOP (P < 0.01) at concentrations of 0.1% and 0.01% but not at concentrations of 0.001% and 0.0001%. There were significant differences among the four concentrations, except for 0.001% versus 0.0001% (P < 0.05, Steel-Dwass test). Therefore, we used 0.01% PGE2 methyl ester for further studies in knockout mice.

IOP Reduction with 0.01% PGE2 Methyl Ester in FPKO, EP1KO, EP2KO, and EP3KO Mice

Single receptor-dependent IOP reductions with 0.01% PGE2 methyl ester were assessed in FPKO, EP1KO, EP2KO, and EP3KO mice and in WT mice. IOP with 0.01% PGE2 methyl ester in FPKO, EP1KO, EP2KO, and EP3KO was measured at 3 hours after application (n = 8 for each genotype). IOP reductions with 0.01% PGE2 methyl ester compared with the carrier in FPKO, EP1KO, EP2KO, EP3KO, and WT mice were 16.7% ± 3.6% (P = 0.0117), 17.6% ± 2.5% (P = 0.0077), 18.3% ± 1.4% (P = 0.0117), 18.0% ± 3.6% (P = 0.0116), and 16.9% ± 2.4% (P = 0.0117), respectively (Wilcoxon signed-rank test). There were no significant differences among FPKO, EP1KO, EP2KO, EP3KO, and WT mice (P = 0.5167, Kruskal-Wallis test; Fig. 3 ).

Effects of Selective EP-Receptor Agonists in WT Mice

IOP reductions with 0.1% of specific EP-receptor agonists (ONO-DI-004, EP1 agonist; ONO-AE1-259-01, EP2 agonist; ONO-AE-248, EP3 agonist; ONO-AE1-329, EP4 agonist) were measured 2 hours after application in WT mice (n = 8). ONO-DI-004 (EP1 agonist), ONO-AE1-259-01 (EP2 agonist), ONO-AE-248 (EP3 agonist), and ONO-AE1-329 (EP4 agonist) lowered IOP by 0.5% ± 1.6% (P = 0.6121), 21.1% ± 4.8% (P = 0.0173), 0.1% ± 3.6% (P = 0.8886), and 17.5% ± 2.9% (P = 0.0117), respectively (Wilcoxon signed-rank test). ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) significantly lowered IOPs of agonist-treated eyes in comparison with those of contralateral carrier–treated eyes, but ONO-DI-004 (EP1 agonist) and ONO-AE-248 (EP3 agonist) had no significant effects (Fig. 4) . Thus, ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) were used for further study.

Time Course and Dose Dependency of IOP Reduction with ONO-AE1-259-01 (EP2 Agonist) and ONO-AE1-329 (EP4 Agonist) in WT Mice

IOP was measured in WT mice at 1, 2, 3, and 6 hours after topical administration of 0.1% ONO-AE1-259-01 (EP2 agonist) and 0.1% ONO-AE1-329 (EP4 agonist). IOP reductions at 1, 2, 3, and 6 hours after administration of 0.1% ONO-AE1-259-01 (EP2 agonist) were 2.1% ± 1.8% (P = 0.2626), 21.1% ± 4.8% (P = 0.0173), 16.4% ± 2.6% (P = 0.0077), and 6.4% ± 4.6% (P = 0.1282), respectively. IOP reductions at 1, 2, 3, and 6 hours after administration of 0.1% ONO-AE1-329 (EP4 agonist) were 17.2% ± 1.8% (P = 0.0117), 17.5% ± 2.9% (P = 0.0117), 15.9% ± 1.8% (P = 0.0117), and 6.0% ± 1.7% (P = 0.0173) (n = 8 at each time point for each agonist; Wilcoxon signed-rank test; Fig. 5 ). Dose-dependent IOP reductions with 0.1%, 0.01%, 0.001%, and 0.0001% ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) were assessed at 2 hours because that was when a 0.1% concentration of each agonist showed maximum IOP reduction. IOP reductions with 0.01%, 0.001%, and 0.0001% ONO-AE1-259-01 (EP2 agonist) were 8.2% ± 4.4% (P = 0.1235), 5.1% ± 3.4% (P = 0.2626), and 3.3% ± 3.2% (P = 0.4838), respectively. IOP reductions with 0.01%, 0.001%, and 0.0001% ONO-AE1-329 (EP4 agonist) were 6.7% ± 1.6% (P = 0.0173), 3.1% ± 1.2% (P = 0.0357), and 2.1% ± 0.8% (P = 0.0117) (n = 8 for each dose and each agonist; Wilcoxon signed-rank test). IOP reductions with 0.1% ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) were significantly greater than those of the other doses of each agonist (P < 0.05, Steel test; Fig. 6 ).

Effects of ONO-AE1-259-01 (EP2 Agonist) and ONO-AE1-329 (EP4 Agonist) on IOP Reduction in Prostanoid-Receptor Knockout Mice

Receptor-dependent IOP reduction by each EP agonist was studied using FPKO, EP1KO, EP2KO, and EP3KO. IOP reductions with 0.1% ONO-AE1-259-01 (EP2 agonist) in FPKO, EP1KO, EP2KO, EP3KO, and WT mice 2 hours after administration were 17.4% ± 4.8% (P = 0.0180), 17.9% ± 1.7% (P = 0.0117), −0.1% ± 3.8% (P = 0.8658), 17.0% ± 4.3% (P = 0.0173), and 21.1% ± 4.8% (P = 0.0117), respectively. IOP reductions with 0.1% ONO-AE1-329 (EP4 agonist) were 15.3% ± 4.3% (P = 0.0077), 14.6% ± 2.1% (P = 0.0117), 16.8% ± 2.7% (P = 0.0180), 10.2% ± 3.4% (P = 0.0180), and 17.5% ± 2.9% (P = 0.0357), respectively. The IOP reduction with ONO-AE1-259-01 (EP2 agonist) in EP2KO mice diminished significantly compared with that in other knockout and WT mice. The IOP reduction with ONO-AE1-329 (EP4 agonist) in EP2KO mice was similar to that in the other knockout and WT mice (Fig. 7) .

Effects of ONO-AE1-259-01 (EP2 Agonist) and ONO-AE1-329 (EP4 Agonist) on Outflow Facility

Total outflow facilities (C_total) in WT mice at 2 hours after administration of 0.1% ONO-AE1-259-01 (EP2 agonist), ONO-AE1-329 (EP4 agonist), and the carrier were 0.0066 ± 0.0003, 0.0070 ± 0.0010 and 0.0065 ± 0.0003 μL/min/mm Hg, respectively (n = 8 for each solution). C_total values at 3 hours after topical administration of 0.005% latanoprost and vehicle were 0.0086 ± 0.0002 and 0.0056 ± 0.0005 μL/min/mm Hg, respectively (n = 8). There was a significant difference between the C_total value after latanoprost and that after carrier (P < 0.01, Mann-Whitney U test; Fig. 8A ). In contrast, there was no significant difference between ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) and the carrier (P > 0.05, Kruskal-Wallis test; Fig. 8B ).

Discussion

In this study, we first confirmed the effect of PGE2 methyl ester on mouse IOP. It induced early transient ocular hypertension and then hypotension. PGE2 causes breakdown of the blood-aqueous barrier.³¹Camras et al.³²demonstrated that topical 0.05% or 0.5% PGE2 administration caused an initial increase followed (3–4 hours later) by a prolonged decrease in rabbit IOP. Although we did not observe obvious signs of intraocular inflammation, such as conjunctival hyperemia, under the operating microscope in mice used in this study, we thought that ocular inflammatory reaction was also responsible for the transient hypertension observed in mouse eyes.

It has been reported that PGE2 has some affinity for the FP receptor (K_i = 100 nM), though it was lower than for the EP receptors (K_i = 20, 12, 0.85, and 1.9 nM for EP1, -2, -3, and -4, respectively).²²IOP reduction with PGE2 methyl ester was similar in FPKO mice and WT mice (Fig. 3) , suggesting that there is another pathway of IOP reduction unrelated to the FP receptor. However, IOP reductions with PGE2 methyl ester in EP1KO, EP2KO, and EP3KO mice were similar to those in WT and FPKO mice. We could not assess PGE2-induced IOP reduction in EP4KO because of embryonic lethality in this strain. When several receptors have common signaling pathways, a single receptor deletion should have little effect on the final pharmacologic action. Because PGE2 binds to a broad spectrum of EP receptor subtypes and FP, even if there is a deficiency of one of the receptors that reduces IOP, other receptors may compensate. Thus, we designed our study to evaluate subtype-specific EP agonists on IOP reduction. All the EP agonists used in this study are free acids but penetrate the anterior chamber after administration (unpublished data, 2008). Given that the penetration of free acids into the anterior chamber is less than that of esters, higher doses of ophthalmic solutions were required to obtain a significant IOP-lowering effect. As described, we found that ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) significantly lowered IOP, but ONO-DI-004 (EP1 agonist) and ONO-AE-248 (EP3 agonist) agonists had no effect (Figs. 4 5 6 7) . Moreover, the effect of ONO-AE1-259-01 (EP2 agonist) was abolished in EP2KO mice. EP2 and EP4, which are Gs-coupling receptors, may share a common intracellular signaling pathway. Thus, the similarity between the effects of PGE2 methyl ester in EP2KO mice and in WT, EP1KO, and EP3KO mice can probably be attributed to its EP4 receptor stimulation (Fig. 1) . This is the first report indicating the possibility that not only EP2 but also EP4 receptors can cause IOP reduction in response to PGE2 methyl ester.

In human ocular tissues, EP1 (epithelium of the cornea, conjunctiva, lens, iris ciliary body, trabecular cells, and retina), EP2 (epithelium, endothelium, and stroma of the cornea; epithelium of the conjunctiva and iris ciliary body), EP3 (epithelium of the cornea; epithelium and stroma of the conjunctiva, iris ciliary body, trabecular cells, and retina), and EP₄ (epithelium, endothelium, and stroma of the cornea and trabecular cells; epithelium and stroma of the conjunctiva and iris ciliary body) receptors have been demonstrated by immunohistochemistry and reverse transcription–polymerase chain reaction.³³ ³⁴ ³⁵ ³⁶ ³⁷In mouse ocular tissues, EP1 (epithelium of the cornea; epithelium of the conjunctiva; nonpigmented epithelium of ciliary body, trabecular meshwork, Schlemm’s canal area, retina), EP2 (epithelium and endothelium of the cornea; epithelium of the conjunctiva, blood vessels, sphincter muscle of the iris; nonpigmented epithelium of ciliary body, trabecular meshwork, Schlemm’s canal area, retina), EP3 (epithelium of the cornea; nonpigmented epithelium of ciliary body), and EP4 (epithelium and endothelium of the cornea; epithelium of the conjunctiva, nonpigmented epithelium of ciliary body, trabecular meshwork, Schlemm’s canal area, retina) receptors have also been demonstrated by immunohistochemistry.³³In addition, it has been reported that the pattern of distribution of EP receptor subtypes in ocular tissues is similar in humans and mice.³³According to a previous report on the expression of EP receptor subtypes in the iris-ciliary body and Schlemm’s canal area, there is abundant expression of EP2, moderate expression of EP4, weak expression of EP1, and negligible expression of EP3.³³

EP1 receptor stimulation increases intracellular Ca²⁺ ³⁸ ³⁹and was originally described as a smooth muscle constrictor.⁴⁰Though latanoprost acid has a relatively high affinity for the EP1 receptor (EC₅₀ = 119 nM),⁴¹we found that latanoprost significantly reduced IOP in EP1KO mice as it did in WT mice.¹⁷Furthermore, there is no previous evidence showing a reduction in IOP with EP1 receptor stimulation. Our study also showed that a specific EP1 agonist (ONO-DI-004) did not significantly reduce IOP. Taken together, these results suggest that EP1 receptor does not play a role in IOP reduction.

In contrast, ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) appear to play an important role in IOP reduction. In addition to reducing IOP, the EP2 and EP4 receptors have a similar intracellular signaling mechanism. Both are coupled primarily to Gs and increase intracellular adenosine 3′, 5′-cyclic monophosphate (cAMP) through the stimulation of adenylate cyclase.⁴²The EP2 receptor has been identified in smooth muscle of vessels from various species and mediates the relaxation of blood vessels.⁴³ ⁴⁴The EP4 receptor has been demonstrated in ductus arteriosus,⁴⁵and venous smooth muscle.⁴⁶Thus, EP4 knockout mice are not available because of patent ductus arteriosus. It has been reported that the EP2 receptor is involved in the relaxation of ciliary muscle cells in culture and may facilitate uveoscleral outflow,⁴⁷and the EP4 receptor causes smooth muscle relaxation.⁴⁶Thus, these receptors may cause ciliary muscle relaxation leading to an increase in uveoscleral outflow.

In addition to the short-term mechanism for increasing uveoscleral outflow by relaxing ciliary muscle, EP2 and EP4 may also have a long-term mechanism for IOP reduction through biological changes of extracellular matrix (ECM). Increases in the biosynthesis of certain matrix metalloproteinases (MMPs) and subsequent remodeling of the extracellular matrix are thought to be critical in the facilitative action of latanoprost on uveoscleral outflow through the ciliary muscle, iris root, and sclera.¹⁵ ⁴⁸ ⁴⁹Upregulation of cyclooxygenase-2 (COX-2) stimulated by latanoprost leads to subsequent MMP-1 expression in human nonpigmented ciliary epithelial cells.¹⁵COX-2 upregulation was also induced by PGE2 through EP2 and EP4 receptors, increasing intracellular cAMP.⁵⁰Additionally, butaprost, an EP2 agonist, significantly increased uveoscleral outflow from the eyes of cynomolgus monkeys after application for 5 days.⁵¹These results suggest that EP2 and EP4 receptor activation may elicit the remodeling of ECM to induce the increase of uveoscleral outflow.

In this study, the C_total value was not affected, at least by a single application, by ONO-AE1-259-01 (EP2 agonist) or ONO-AE1-329 (EP4 agonist). The C_total value measured by the two-level, constant-pressure perfusion method²⁶ ²⁷ ²⁸is thought to represent status in the pressure-dependent outflow system (conventional outflow route).²⁸ ²⁹ ³⁰Therefore, our results suggest that ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) reduce mouse IOP mainly by the increase of the pressure-independent outflow system (uveoscleral outflow) or the decrease of aqueous humor production, or both. Because the measurement of aqueous flow volume is difficult in small mouse eyes, additional studies using monkey eyes will be needed to further clarify the mechanism of IOP reduction induced by EP2 and EP4 receptor. On the other hand, C_total was significantly increased in mouse eyes by a single instillation of latanoprost, suggesting that acute IOP reduction by a single instillation of latanoprost in mouse eyes was attributable to an increase in pressure-dependent outflow.

A single application of EP3 agonist as used in this study revealed no effect on mouse IOP. In the iris-ciliary body and Schlemm’s canal area of human and mouse eyes, EP3 was expressed least among the four EP receptor subtypes.³³The EP3 receptor, contrary to the EP2 receptor, is believed to inhibit adenylate cyclase mainly through Gi⁵² ⁵³ ⁵⁴and to mediate smooth muscle cell contraction.⁵⁵Moreover, EP1 and EP3 neither increase cAMP nor induce COX-2, in contrast to EP2 and EP4.⁵⁰Thus, because EP3 may have an inverse signal to EP2 and EP4, the lack of IOP reduction with EP3 is a credible result.

Besides ocular hypotensive effects, subtypes of EP receptors can mediate other untoward effects. For example, a relationship between PGE2 and angiogenesis has been reported.⁵⁶It has also been reported that PGE2 induced by COX-2 increased vascular endothelial growth factor and caused angiogenesis. Several investigators have shown that all subtypes of EP receptors could mediate neovascularization induced by PGE2.⁵⁷ ⁵⁸Thus, it is necessary to take these pharmacologic actions into account when we use prostanoid EP receptor subtype agonists in clinical applications.

Although the results obtained in mouse eyes may not be directly extrapolated to human eyes, the screening of chemical compounds and the development of new antiglaucoma medicine in mouse eyes deserves consideration because IOP reduction in mouse eyes by a commercially available PG analogue is similar to that in human eyes.²⁵

In conclusion, we demonstrated IOP-lowering effects of PGE2 methyl ester, ONO-AE1-259-01 (EP2 agonist), and ONO-AE1-329 (EP4 agonist) in mouse eyes. In addition to IOP reduction in response to FP receptor stimulation with available PG analogues,⁷ ⁸it was found that EP2 and EP4 may play an important role in IOP reduction. The mechanisms of IOP reduction by ONO-AE1-259-01 (EP2 agonist) or ONO-AE1-329 (EP4 agonist) are dependent on EP2 receptor and EP4 receptor, respectively; these agonists have cellular signaling mechanisms different from those of FP agonists. No contribution of EP1 or EP3 receptors to IOP reduction was evident in this study.

Supported in part by Grants H18-Kankakuippan-001 from the Ministry of Health, Labor, and Welfare of Japan, and A18209053 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MAr).

Submitted for publication August 31, 2008; revised November 8 and December 11, 2008; accepted March 16, 2009.

Disclosure: T. Saeki, None; T. Ota, None; M. Aihara, None; M. Araie, None

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: Makoto Aihara, Department of Ophthalmology, University of Tokyo School of Medicine, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan; [email protected].

Figure 1.

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Time course of 0.1% PGE2 methyl ester–induced IOP reduction in WT mice. Ophthalmic solution was applied at 6:00 PM, and IOP was measured 1, 2, 3, and 6 hours later with a microneedle. Data are expressed as mean ± SEM (n = 10/time point). *P < 0.01 for treated versus contralateral carrier–treated eye (Wilcoxon signed-rank test).

Figure 2.

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Dose-dependent IOP reduction by PGE2 methyl ester in WT mice. PGE2 methyl ester at each dose was instilled at 6:00 PM, and IOP was measured 3 hours later. Data are expressed as mean ± SEM (n = 8 for each dose). *P < 0.012 for treated versus contralateral carrier–treated eye (Wilcoxon signed-rank test).

Figure 3.

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IOP-lowering effect of 0.01% PGE2 methyl ester in FPKO, EP1–3KO, and WT mice. IOP was measured at 3 hours after administration. Data are expressed as mean ± SEM (n = 8 for each genotype). In all genotypes, 0.01% PGE2 methyl ester showed statistically significant IOP reduction compared with the carrier (P < 0.012, Wilcoxon signed rank test). There was no significant difference in IOP reduction among the four types of knockout (KO) mice and WT mice. P = 0.5167 (Kruskal-Wallis test).

Figure 4.

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IOP-lowering effects of 0.1% ONO-DI-004 (EP1 agonist), ONO-AE1-259-01 (EP2 agonist), ONO-AE-248 (EP3 agonist), and ONO-AE1-329 (EP4 agonist). Each drug was applied at 6:00 PM, and IOP was measured at 2 hours after administration. Data are expressed as mean ± SEM (n = 8 for each agonist). *P < 0.02 for treated versus contralateral carrier–treated eye (Wilcoxon signed-rank test).

Figure 5.

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Time course of IOP reduction with 0.1% ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) in WT mice. Data are expressed as mean ± SEM (n = 8). *P < 0.02 for treated versus contralateral carrier–treated eye (Wilcoxon signed rank test).

Figure 6.

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Dose-dependent IOP reduction with ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) in WT mice. Data are expressed as mean ± SEM (n = 8 for each dose). IOP was measured at 2 hours after administration. *P < 0.05 for treated versus contralateral carrier–treated eye (Wilcoxon signed-rank test). #P < 0.05 compared 0.1% with 0.01% or lower concentrations of each agonist (Steel test).

Figure 7.

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IOP reduction with 0.1% ONO-AE1-259-01 (EP2 agonist) and ONO-AE1-329 (EP4 agonist) in FPKO, EP1–3KO, and WT mice. IOP was measured at 2 hours after administration. Data are expressed as mean ± SEM (n = 8 in each genotype). *P < 0.05 for treated versus contralateral carrier–treated eye (Wilcoxon signed-rank test).

Figure 8.

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Effects of ONO-AE1-259-01 (EP2 agonist), ONO-AE1-329 (EP4 agonist), and latanoprost on outflow facility. C_total value indicates total outflow facility measured by the two-level constant-pressure perfusion method. (A) C_total values with 0.005% latanoprost and its carrier solution. *P < 0.01 by Mann-Whitney U test. (B) C_total values with 0.1% ONO-AE1-259-01 (EP2 agonist), 0.1% ONO-AE1-329 (EP4 agonist), and its carrier solution. Data are expressed as mean ± SEM (n = 8 in each solution).

The authors thank Ono Pharmaceutical (Osaka, Japan) for kindly providing the EP receptor agonists, and Shuh Narumiya (Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan) for kindly providing the prostanoid receptor knockout mice.

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