Responses of Intraocular Pressure and the Pupil of Feline Eyes to Prostaglandin EP₁ and FP Receptor Agonists

Abstract

purpose. Previous studies suggested that FP receptors do not mediate the relaxation of the ciliary muscle and reduction of intraocular pressure in cats by prostaglandin (PG) F_2α. The present study was undertaken to determine whether the reduction of intraocular pressure in cats induced by PGF_2α is mediated by FP or other prostaglandin receptors.

methods. One eye of each cat was treated topically with prostaglandin F_2α, fluprostenol (FP receptor agonist), or 17-phenyl trinor PGE₂ (EP₁ receptor agonist) in a dose range of 12.5 to 50 μg. The effects of SC19220 and SC51089 (EP₁ receptor antagonists), BWA868c, and SQ29548 (DP and TP receptor antagonists, respectively) on the intraocular response to PGF_2α were also examined. At intervals up to 6 hours after treatment, intraocular pressure was measured with a pneumotonometer, and pupil diameters were measured with a millimeter ruler.

results. In the dose ranges used, PGF_2α and 17-phenyl trinor PGE₂ decreased intraocular pressure and pupil diameter. The greatest reduction of intraocular pressure by 50.0 μg PGF_2α was 5.0 ± 1.4 mm Hg, whereas that by 50 μg 17-phenyl trinor PGE₂ was 6.2 ± 1.5 mm Hg. The isopropyl ester of PGF _2α at a dose of 1.25 μg reduced intraocular pressure by 3.75 ± 0.25 mm Hg at 2 hours. At doses up to100 μg, fluprostenol did not decrease intraocular pressure but did reduce pupil diameter. SC19220, a weak but selective EP₁ receptor antagonist, inhibited the intraocular pressure response to both PGF_2α and 17-phenyl trinor PGE₂. The more potent EP₁ receptor antagonist SC51089 had a greater inhibitory effect than SC19220 on the intraocular pressure response to PGF_2α. Both of these antagonists had a small but non–dose dependent and statistically insignificant effect on the pupil response to PGF_2α. These observations suggest that in cats, intraocular pressure and pupil responses to PGF_2α are mediated by EP₁ and FP receptors, respectively. However, SC19220 significantly and dose-dependently inhibited the pupil response to 17-phenyl trinor PGE₂, suggesting that EP₁ receptors mediate pupil response to this agonist. DP and TP receptor antagonists at doses 5- to 20-fold greater than the IC₅₀ values had no effect on the ocular hypotensive response to PGF_2α. The concurrent administration of 12.5μ g of each of PGF_2α and 17-phenyl trinor PGE₂ did not produce an additive effect on intraocular pressure, indicating that in cats PGF_2α and 17-phenyl trinor PGE₂ act on the same receptor type.

conclusions. These results suggest that a significant proportion of the ocular hypotensive action of PGF_2α in cats is mediated by EP₁ but not by FP receptor. Evidence was also provided to show that 17-phenyl trinor PGE₂ is an ocular hypotensive agent in cats.

Prostaglandins (PGs), administered topically in appropriate concentrations, reduce intraocular pressure (IOP) in rabbits, cats, and humans. ^{1 2 3 4} Prostaglandin F_2α in particular is an extremely potent ocular hypotensive agent and is currently thought to reduce intraocular pressure by increasing uveoscleral outflow, ^{5 6} perhaps through the mediation of discrete FP receptors in the ciliary muscle. ^{7 8 9 10} However, one report suggests that the ocular hypotensive action of PGF_2α in the cat is not mediated by FP receptors. ¹¹ Also, PGF_2α has been reported to have a weak relaxant effect on cat ciliary muscle, whereas the more selective FP receptor agonist 17-phenyl trinor PGF_2α was without effect. ^{12 13} These observations suggest that increased uveoscleral outflow in response to PGF_2α may not be mediated by FP receptors. Recently, a study on the generation of second messengers reported that 17-phenyl trinor PGE₂, but not PGF_2α, stimulated the formation of inositol phosphates in cat ciliary muscles. ¹⁴ To examine whether the ocular hypotensive action of PGF_2α is mediated by EP₁ or other PG receptors, we investigated the response of IOP and pupil diameter in cats to PGF_2α and fluprostenol, FP receptor agonists, and an EP₁ receptor agonist, 17-phenyl trinor PGE₂. 

Methods

Materials

All natural and synthetic prostaglandins and SQ29548 were purchased from Cayman Chemical (Ann Arbor, MI). The EP₁ receptor antagonists SC19220 and SC51089 were a gift from Searle Research and Development (Skokie, IL). DP receptor antagonist BWA868c was supplied by Glaxo–Wellcome (Hertfordshire, UK). Alcaine (0.5% proparacaine) was purchased from Alcon Laboratories (Ft. Worth, TX). 

Measurement of IOP and Pupil Diameter

All animals used were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty cats (2–3 kg) were domesticated for 4 months and trained to accept topical administration of saline or local anesthetic drops and the application of a pneumotonometer tip to the cornea to measure IOP. These cats were randomly divided into groups of three to six cats depending on the experimental design of each experiment. A washout period of 15 to 30 days was allowed between separate experiments. Before the experiments with prostaglandins, IOPs of the eyes treated with the vehicle and untreated contralateral eyes of all the cats were measured for a 6-hour period using a pneumotonometer (model 30-Classic; Mentor O & O, Norwell, MA). This pneumotonometer was calibrated for human eyes by the manufacturer and has been used to measure IOP of cats ^{15 16 17} without further calibration. We did not calibrate it for cat eyes because our study is not concerned with the physiology or comparative physiology of IOP. In our pharmacological study, we were interested in the effects of prostaglandins on IOP and the differences between the treated and the control eyes. Intraocular pressure measured in cats using this pneumotonometer in the present study was in the range of 17 to 19 mm Hg compared with 14 to 20, ¹⁵ 17 to 20, ¹⁶ and 19 to 20 mm Hg ¹⁷ as reported previously. 

Prostaglandins, diluted in dimethyl sulfoxide (DMSO), were instilled onto the cornea of one eye in a 25-μl volume; the contralateral eye received the same volume of DMSO alone. In experiments with antagonists, eyes were pretreated 30 minutes before PGF_2α with an appropriate antagonist. This time of pretreatment with the antagonists was determined in our preliminary studies with SC19220, in which it was observed that 10 to 30 minutes’ pretreatment was satisfactory to exert its inhibitory effect. Intraocular pressure was measured at 1 and 2 hours before and at 0, 1, 2, 4, and 6 hours after treatment with prostaglandin receptor agonists and at the same time of the day beginning at 9:00 AM. Before the measurement of IOP, a drop of local anesthetic, proparacaine hydrochloride (0.5%), was instilled onto the cornea. Horizontal pupil diameter before and after treatment was measured with a millimeter ruler. 

Expression and Analysis of Data

Both the IOP and pupil diameter data were expressed as the difference between drug-treated eyes and DMSO-treated contralateral eyes. The significance of the difference between the mean values of DMSO- and drug-treated eyes at any given time was evaluated by an ANOVA test using Microsoft Excel 5.0. The IC₅₀ values were calculated using GraphPad Prizm (version 2.01; GraphPad Software, San Diego, CA) and Lotus 123-R4.01. 

Results

The effects of PGF_2α, fluprostenol, and 17-phenyl trinor PGE₂ on IOP and the pupil diameters of cats are summarized in Figures 1 and 3 . In the dose range of 12.5 to 50.0 μg, PGF_2α reduced IOP without an initial phase of ocular hypertension. All doses of PGF_2α lowered IOP, beginning 1 hour after the treatment. The greatest reduction, 5.0 ± 0.4 mm Hg, occurred between 1 and 2 hours after treatment with 50.0 μg of PGF_2α (Fig. 1A) . The IOP returned to baseline value within 6 hours after treatment with all doses of PGF_2α. In contrast, the response of IOP to 1.25 μg isopropyl ester of PGF_2α was significantly greater than that to PGF_2α (Fig. 1A) . This was not unexpected because the ester forms of prostaglandins achieve greater intraocular concentrations than do their acidic forms. ¹⁸ 17-Phenyl trinor PGE₂ also reduced intraocular pressure in a dose-dependent manner, with an onset and duration of action similar to those of PGF_2α (Fig. 1B) . The greatest reduction of IOP by 50.0 μg of 17-phenyl trinor PGE₂ was 6.2 ± 1.5 mm Hg at 2 hours after treatment. Fluprostenol, in doses up to 100 μg, did not reduce IOP (Fig. 1C) . Intraocular pressures of the eyes treated with vehicle or 100 μg SC19220 and the contralateral eyes of the 50 μg PGF_2α–treated group are shown in Figure 2 . It is obvious that feline IOP remains stable during a 6-hour experimental period with minor variations. At the maximal concentration, none of the agonists induced a contralateral effect. Furthermore, SC19220 alone at 100 μg had no effect on IOP. 

Prostaglandin F_2α, its isopropyl ester 17-phenyl trinor PGE₂, and fluprostenol all dose-dependently induced miosis. The time course and duration of the pupil response to these agonists were similar to those of the IOP responses (Figs. 3 A, 3B, and 3C). As shown in Table 1 , the selective EP₁ receptor antagonist SC19220 inhibited the IOP response to PGF_2α or 17-phenyl trinor PGE₂ as well as isopropyl PGF_2α. The highest concentration of SC19220 blocked the IOP response to PGF_2α by 38%, compared with the 80% and 94% reductions of the responses to17-phenyl trinor PGE₂ and isopropyl PGF_2α, respectively. SC51089, a more potent EP₁ receptor antagonist than SC19220, confirmed the above observation on PGF_2α response. The results (Table 1) show that SC51089 is more effective than SC19220 in inhibiting the IOP response to PGF_2α. Inhibition of the pupil response to PGF_2α by SC19220 or SC51089 was not dose-dependent or statistically insignificant; whereas the inhibition of 17-phenyl trinor PGE₂ response by SC19220 was dose-dependent and significant (SC51089 was not tested against 17-phenyl trinor PGE₂). The highly potent DP receptor antagonist BWA868c (IC₅₀, 1 nM) at 0.3 ng (25 nM) or 1.2ng (100nM) and TP receptor antagonist SQ29548 (IC₅₀, 10 nM) at 0.48 ng (50 nM) or 0.96 ng (100 nM) did not affect the ocular hypotensive response to PGF_2α (Table 1) . We performed one classic pharmacological experiment to determine whether PGF_2α and 17-phenyl trinor PGE₂ are acting on the same or different receptor type. Figure 4 shows that 12.5 μg of each of these agonists administered together did not produce a greater hypotensive effect than when administered alone. This observation suggests that these two agonists activated the same EP₁ receptors. If they had been acting on different receptor types, the IOP response would have been additive. 

Discussion

In the present studies, we demonstrated for the first time that a single dose of 17-phenyl trinor PGE₂, an EP₁ receptor agonist, reduced IOP and pupil diameter in cats. Prostaglandin F_2α also reduced IOP and pupil diameter in a dose-dependent manner. At a low dose of 1.25 μg, the isopropyl ester of PGF_2α also reduced IOP. To maintain consistency, we primarily used prostaglandins instead of their isopropyl esters because two of the agonists used, fluprostenol and 17-phenyl trinor PGE₂, are not available as isopropyl esters. 

Intraocular pressure responses to PGF_2α and its isopropyl ester are consistent with previous reports that these compounds are potent ocular hypotensive and miotic agents in cats. ^{2 18 19} Fluprostenol, a more selective FP receptor agonist than PGF_2α, did not lower IOP but was as potent as PGF_2α and 17-phenyl trinor PGE₂ as a miotic agent. The EP₁ receptor antagonist SC19220 at a 100-μg dose significantly inhibited the reduction in IOP by PGF_2α, its isopropyl ester, and 17-phenyl trinor PGE₂. The doses of the antagonist used in the present study appear to be high. However, it should be emphasized that, although this EP₁ antagonist is highly selective, it is not potent, particularly in vivo. Therefore, it is not surprising that high doses were needed to exert an inhibitory effect. The more potent EP₁ receptor antagonist SC51089, at a dose of only 10 μg, inhibited 80% of the IOP response to PGF_2α, suggesting that the ocular hypotensive action of PGF_2x is mediated by EP₁ but not by FP receptors in the cat. These observations were further supported by the fact that fluprostenol, a more selective FP receptor agonist than PGF_2α, did not lower IOP while contracting the sphincter muscle to induce miosis. If FP receptors were present in the ciliary body, fluprostenol would have reduced IOP as it contracted the sphincter muscles that are known to express FP receptors. Furthermore, the observation that the concurrent administration of PGF_2α and 17-phenyl trinor PGE₂ was not additive in lowering IOP indicated that these two agonists were acting primarily on the same EP₁ receptors. If these two agonists were acting on separate receptor types, then the response to the combined treatment would have been greater than the response to either of the agonists alone. The order of potency of prostaglandins for EP₁ receptors is PGE₂ > PGF_2α, prostacyclin > PGD₂ and TXA₂. ²⁰ Therefore, in tissues in which FP receptors are lacking, PGF_2α is most likely to activate EP₁ receptors. Our pharmacological study in vivo only suggests that PGF_2α is acting via EP₁ receptors. To determine whether this is precisely the situation, additional in vitro studies on the contractile or relaxing response of the feline ciliary muscles to FP and EP₁ receptor agonists are needed. 

Ligand binding assays and functional studies suggest that PGF_2α has affinity for DP and EP₃ receptors. ^{21 22 23} Also, stimulation of DP or EP₃ receptors reduces IOP in rabbits, ^{22 23} and stimulation of TP receptors reduces IOP in beagle dogs. ²⁴ Thus, to exclude the possibility that the IOP-lowering effect of PGF_2α was due to the stimulation of either EP₃ or DP receptors or both, we tested a potent and selective DP receptor antagonist, BWA868c. ²⁵ At 25- to 100-fold greater concentration than its IC₅₀, the DP receptor antagonist did not significantly modify PGF_2α response, suggesting that DP receptors are not involved in the ocular hypotension induced by PGF_2α. Although PGF_2α has a poor affinity for TP receptors, to rule out the extreme possibility that PGF_2α also stimulated these receptors, the effect of the TP receptor antagonist SQ29548 ²⁶ on the responses to PGF_2α was examined. Even at high concentrations, this antagonist did not block PGF_2α response. We could not test the effect of an EP₃ receptor antagonist on PGF_2α response because no such antagonists are available. Therefore, we suggest that PGF_2α does not stimulate DP or TP receptors to lower IOP in cats. 

Previous studies have suggested that the reduction of IOP and relaxation of ciliary muscle of cats by PGF_2α are not mediated by FP receptors. ^{11 12 13} Also, it has been reported that PGF_2α does not increase inositol phosphate turnover in the feline ciliary muscles ex vivo. ¹⁴ All these studies suggest a lack of FP receptors in the feline ciliary body but do not describe prostaglandin receptors in cats that are activated by PGF_2α to lower IOP. The data we obtained with fluprostenol, EP₁ receptor antagonists, and concurrent administration of PGF_2α and 17-phenyl trinor PGE₂ suggest that FP receptors are not expressed in the feline ciliary muscles. However, studies on the expression of mRNA of FP receptors in the target tissues, such as the feline ciliary muscles, need to be performed to confirm our pharmacological observations. 

The concentrations of prostaglandins used in the present study appear to be high, and it could be argued that other prostaglandin receptors, particularly EP₁ receptors, were stimulated by such high concentrations. In fact, the doses of prostaglandins used in the present study were smaller than those used in previous studies. ^{2 11 18} It should also be pointed out that a dose of a compound administered topically to the eye is diluted by tears and that only a fraction of the dilution permeates the cornea. Thus, the intraocular prostaglandin concentration will probably range only from 0.25% to 0.5% of the administered dose. ^{27 28 29} Therefore, the intraocular concentrations of the agonists at the doses used in the present study were probably in the range of 0.03 to 0.25μ g. Furthermore, the actual concentration of the agonist in the tissues of the uveoscleral pathway or the site of action of PGF_2α or 17-phenyl trinor PGE₂ is likely to be less than the total concentration in the intraocular tissues. Therefore, prostaglandin agonists, administered topically, most likely stimulated the receptors for which the agonist has primary affinity. This suggestion is supported by the fact that fluprostenol, at large doses of 25 to 50μ g, stimulated only FP receptors in the iris sphincter muscle, not EP₁ or other prostaglandin receptors. 

At present, the mechanism of the ocular hypotensive action of the EP₁ receptor agonist 17-phenyl trinor PGE₂ on IOP is not known. In a recent study, Krauss et al. ³⁰ observed that novel TP receptor agonists reduce IOP and increase aqueous humor outflow in dogs. TP receptor activation results in the increased turnover of inositol trisphosphates followed by mobilization of intracellular calcium, leading to the contraction of smooth muscle cells in the trabecular meshwork and ciliary muscles. These events may be the underlying mechanism of facility increase by TP receptor agonists. Stimulation of EP₁ receptors also mobilizes intracellular calcium, thus it can be speculated that 17-phenyl trinor PGE₂ has the same mechanism of action as TP receptor agonists in lowering IOP. However, such speculation needs to be explored in studies on aqueous humor dynamics. 

 Supported by NIH/NEI Grant EY-06918, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness. Christopher A. Paterson is a Research to Prevent Blindness Senior Scientific Investigator.

 Submitted for publication July 28, 1998; revised January 28, April 27, and June 10, 1999; accepted June 24, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

The reduction of IOP in cats by a single dose of topically administered prostaglandin F_2α, 17-phenyl trinor PGE₂, and fluprostenol. The IOP values are expressed as the net of control minus treated. Each experiment at each concentration was done on three animals, and each experiment was done at least twice. Vertical bars are± SEM. *Significant difference between controls and treated at P < 0.05 level.

Figure 2.

 

Intraocular pressure of vehicle-treated eyes (A; n = 8) and contralateral eyes (B; ▪) of 50 μg PGF_2α–treated groups (n = 8) and IOP of 100 μg SC19220–treated eyes (▾; n = 4). Vertical bars are ±SEM.

Figure 3.

 

The reduction of pupil diameter in cats by PGF_2α (A), 17-phenyl trinor PGE₂ (B), and fluprostenol (C) administered topically. The values are the net of those for control minus treated eyes. Each experiment at each concentration used three animals, and each experiment was done at least twice. Vertical bars are ±SEM. *Significant difference between controls and treated at P < 0.05 level.

Table 1.

 

IC₅₀ Values of Prostaglandin Receptor Antagonists, SC19220 (EP₁), SC51089 (EP₁), BWA868c (DP), and SQ29548 (TP), for the Inhibition of IOP and Pupil Response to PGF_2α and 17-Phenyl Trinor PGE₂ in Cats

Agonist	Antagonist	IOP Decrease (mmHg)*	Pupil Diameter Decrease (mm)*
50 μg PGF2α	0	5.0 ± 1.4 (4)	5.0 ± 1.5 (4)
	SC19220 (μg)
	25	4.2 ± 0.4 (8)	3.3 ± 1.0 (8)
	50	3.5 ± 0.4 (8)	3.6 ± 0.7 (8)
	100	3.1 ± 1.6 (8), †	4.0 ± 0.4 (8)
	SC51089 (μg)
	0	6.3 ± 0.3 (4)	5.75 ± 1.2 (4)
	2.5	6.3 ± 0.4 (4)	4.8 ± 0.9 (4)
	5	2.3 ± 0.4 (4), †	4.9 ± 0.6 (4)
	10	1.3 ± 1.3 (4), †	4.8 ± 1.0 (4)
	BWA868c (ng)
	0	4.5 ± 1.0 (4)	4.2 ± 1.1 (4)
	0.3	4.2 ± 0.5 (4)	2.7 ± 1.3 (4)
	1.2	3.7 ± 0.2 (4)	2.7 ± 0.3 (4)
	SQ29548 (ng)
	0	4.3 ± 0.6 (4)	4.1 ± 0.7 (4)
	0.48	3.7 ± 0.6 (4)	3.3 ± 1.1 (4)
	0.96	3.3 ± 0.6 (4)	3.3 ± 1.4 (4)
	SC19220 (μg)
1.25 μg PGF2α (isopropyl ester)	0	3.5 ± 0.1 (5)	5.2 ± 0.6 (5)
	100	0.2 ± 0.1 (5), †	3.6 ± 0.7 (5)
50 μg 17-phenyl trinor PGE2	0	6.2 ± 1.5 (8)	3.5 ± 0.8 (8)
	25	5.8 ± 0.9 (8)	3.3 ± 0.1 (8)
	50	5.0 ± 1.2 (8)	2.0 ± 1.4 (8)
	100	1.3 ± 1.1 (8), †	0.5 ± 0.4 (8), †

 Decreases in IOP and pupil diameter were measured 2 hours after administration of the agonist and antagonist.

*  Values are mean ± SEM of (n) number of experiments.

†  Significantly different from antagonist untreated eye at P < 0.05 level.

Figure 4.

 

The response of IOP to 12.5 μg of PGF_2α and 17-phenyl trinor PGE₂ administered alone or in combination. The IOP values are the net of those for control minus treated eyes. Each experiment consisted of six animals. Vertical bars are ±SEM.