Human Trabecular Meshwork Cell Responses Induced by Bimatoprost, Travoprost, Unoprostone, and other FP Prostaglandin Receptor Agonist Analogues

Abstract

purpose. To determine the functional agonist potencies of the intraocular pressure (IOP)-lowering prostaglandin F (FP)-class prostaglandin (PG) analogues (e.g., travoprost, latanoprost, bimatoprost, and unoprostone isopropyl ester) in human trabecular meshwork (h-TM) cells, by using phosphoinositide (PI) turnover and intracellular Ca²⁺ ([Ca²⁺]_i) mobilization, and to confirm the FP nature of these receptors by using an FP receptor antagonist, 11β-fluoro-15-epi-15-indanyl-PGF_2α (AL-8810).

methods. FP-receptor-mediated PI turnover and [Ca²⁺]_i mobilization were measured in h-TM cells by determining the accumulation of [³H]-inositol phosphates ([³H]-IPs) by anion-exchange chromatography and real-time fluorescence imaging, respectively.

results. Various PG analogues concentration-dependently stimulated production of [³H]-IPs in h-TM cells with the following agonist potencies (median effective concentration; EC₅₀): travoprost acid (EC₅₀ = 2.4 nM) > cloprostenol (EC₅₀ = 4.5 nM) > (±)-fluprostenol (EC₅₀ = 10.8 nM) > latanoprost acid (EC₅₀ = 34.7 nM) > bimatoprost acid (EC₅₀ = 112 nM) > PGF_2α (EC₅₀ = 120 nM) ≫ unoprostone (UF-021; EC₅₀ = 3280 nM) > S-1033 (EC₅₀ = 4570 nM; all n = 3–9). Prodrug derivatives of these compounds exhibited the following potencies: travoprost (isopropyl ester; EC₅₀ = 89.1 nM) > latanoprost (isopropyl ester; EC₅₀ = 778 nM) > bimatoprost (amide; EC₅₀ = 1410–6940 nM). Travoprost acid, PGF_2α, unoprostone, and S-1033 were tested in addition for [Ca²⁺]_i mobilization and found to have rapid and dose-dependent effects. The FP receptor-selective antagonist AL-8810 antagonized the (±)-fluprostenol-induced PI turnover in these cells (K _i = 2.56 ± 0.62 μM) as well as that induced by bimatoprost and acids of latanoprost and travoprost. The agonist and antagonist potencies of the PG analogues from the PI turnover assays in h-TM cells correlated well with PI turnover data obtained from the cloned human ciliary body FP receptor (r = 0.92; P < 0.0001).

conclusions. The pharmacology of the h-TM cell FP-receptor-mediated PI turnover and [Ca²⁺]_i mobilization was defined using numerous synthetic (FP-selective) PG agonist analogues and an FP receptor antagonist, AL-8810. Bimatoprost, travoprost, latanoprost, unoprostone isopropyl ester, and their respective free acids were shown to be FP agonists in the h-TM cells.

Isopropyl ester prodrugs of prostaglandin F (FP)-class prostaglandin (PG) receptor agonists, including travoprost, ¹ latanoprost, ² and unoprostone isopropyl ester ³ lower intraocular pressure (IOP) in a number of mammalian species, including humans, and are used to treat ocular hypertension and glaucoma. ⁴ Another prostaglandin analogue prodrug, bimatoprost (17-phenyl-trinor PGF_2α ethyl amide), ⁵ has also recently been marketed for this indication. Even though putative FP receptors have been detected in the human ciliary muscle ^{6 7 8} and human trabecular meshwork (h-TM) ⁹ cells, and an FP receptor from human ciliary body cDNA has been cloned, ¹⁰ the detailed pharmacologic characterization of the FP-receptor-mediated functional responses in these human ocular tissues and cells has not been described to date. In view of the paucity of this type of pharmacologic information, the purposes of our current studies were to determine the pharmacologic properties of functionally coupled FP receptors in h-TM cells derived from several human donors without glaucoma, by using selective FP receptor agonist prodrugs such as latanoprost and travoprost and their respective free acids; to assess the ability of some of these FP receptor agonists to mobilize intracellular Ca²⁺ ([Ca²⁺]_i) in h-TM cells; and to determine the antagonist effects of a novel FP-receptor antagonist 11β-fluoro-15-epi-15-indanyl-PGF_2α (AL-8810) ¹¹ at the h-TM FP receptors, to complete the characterization of these receptors. To our knowledge, this represents the first such detailed study of FP receptor pharmacology in h-TM cells expressing endogenous FP prostaglandin receptors. 

Materials and Methods

The reagents were obtained from the cited sources as follows: all tissue culture media, antibiotics, supplements, and trypsin-EDTA from Life Technologies (Grand Island, NY); fetal bovine serum from Hyclone (Logan, UT); formic acid, ammonium formate, LiCl, and type B gelatin from Sigma Chemical Co. (St. Louis, MO); [³H]-myo-inositol from Amersham Corp. (Deerfield, IL); AG-1X8 anion-exchange resin from Bio-Rad (Hercules, CA); scintillation fluid (Ecolume) from ICN Biomedicals (Costa Mesa, CA); and bimatoprost, bimatoprost acid, (±)-fluprostenol (1:1 mixture of [+]) and [−] enantiomers), latanoprost, unoprostone, and unoprostone isopropyl ester from Cayman Chemical Co. (Ann Arbor, MI). Travoprost ([+]-fluprostenol isopropyl ester), travoprost free acid ([+]), latanoprost acid, and AL-8810 were synthesized in the Medicinal Chemistry Department of Alcon Research, Ltd. (Fort Worth, TX). Bimatoprost (Lumigan) was from Allergan, Inc. (Irvine, CA). S-1033 was generously provided by Shionogi (Osaka, Japan). FLIPR and the Ca²⁺-sensitive dye kit were purchased from Molecular Devices Corp. (Menlo Park, CA). The h-TM cells were kindly provided by Mari Engler, Therapeutic Target Research (Alcon Research, Ltd). 

Cell Culture

The h-TM cells were obtained as previously described ¹² from dissected TM explants of human donor eyes (from six different donors, ages 0.5, 44, 51, 54, 80, and 85 years; all patients with no ocular disease history) kindly provided by various Eye Banks in the United States. The identity of h-TM cells isolated from these explants was confirmed by a battery of biochemical and immunohistochemical techniques. ¹² The h-TM cells were grown in DMEM with 1 g/L glucose supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine, and 10% fetal bovine serum. When confluent, these cells were subcultured and seeded into uncoated 24-well plates for the phosphoinositide (PI)-turnover experiments described. Cells were maintained in a humidified atmosphere of 5% CO₂ and 95% air, with two changes of fresh medium weekly. Cells from passages 1 to 8 were used in the studies. 

PI Turnover Assay

PI turnover assays of phospholipase C activity were conducted as previously described and involved the measurement of agonist-stimulated production of [³H]-inositol phosphates ([³H]-IPs) by anion-exchange chromatography. ^{13 14} Briefly, confluent h-TM cells (∼50,000/well) were exposed for 24 to 30 hours to 3.8 μCi [³H]-myo-inositol (18.3 Ci/mmol) in 1.0 mL of the respective serum-free medium to label the cell membrane phospholipids. Cells were then rinsed once with DMEM/Ham’s F-12 containing 10 mM LiCl before incubation with the agonist (or solvent as the control) in 1.0 mL of the same medium for 1 hour at 37°C, after which the medium was aspirated and 1 mL cold 0.1 M formic acid was added. When the antagonist effects of AL-8810 were studied, it was added to the cells 15 minutes before exposure to the agonist and the assay continued for another hour in the presence of the antagonist. The chromatographic separation of [³H]-IPs on an AG-1X8 resin-containing column was performed as previously described, ^{13 14} with sequential washes with water and 50 mM ammonium formate, followed by elution of the total [³H]-IPs fraction with 1.2 M ammonium formate containing 0.1 M formic acid. The eluate (4 mL) was collected and mixed with 15 mL scintillation fluid, and the total [³H]-IPs were determined by scintillation counting on a beta-counter at ∼50% efficiency (LS6000; Beckman Instruments, Carlsbad, CA). 

Intracellular Ca²⁺ Mobilization Assay

FP receptor-mediated mobilization of intracellular Ca²⁺ ([Ca²⁺]_i) was studied in h-TM cells with a fluorometric imaging plate reader (FLIPR; Molecular Devices Corp.), as previously described. ^{15 16} In brief, h-TM cells were transferred in a 50-μL volume at a density of 50,000 cells per well to black-walled, 96-well tissue culture plates and cultured for two more days, to allow the cells to attach and flatten out in the culture plates. On the day of the experiment, one vial of FLIPR dye (Calcium Assay Kit; Molecular Devices Corp.) was resuspended in 50 mL of an FLIPR buffer consisting of Hanks’ balanced salt solution (HBSS), 20 mM HEPES buffer, and 2.5 mM probenecid (pH 7.4). The h-TM cells were then loaded with the calcium-sensitive dye by addition of an equal volume (50 μL) to each well of the 96-well plate and incubated with the dye for 1 hour at 23°C. The compound plate and cell plate were then placed in the FLIPR instrument. At the beginning of an experimental run, a signal test was performed to check the basal fluorescence signal from the dye-loaded cells and the uniformity of the signal across the plate. The basal fluorescence was adjusted to between 8,000 and 12,000 units by modifying the exposure time, the camera F-stop, or the laser power. Instrument settings for a typical assay were the following: laser power 0.3 to 0.6 W, camera F-stop F/2, and exposure time 0.4 seconds. An aliquot (25 μL) of the FP receptor agonist was then added to the existing 100 μL dye-loaded cells at a dispensing speed of 50 μL/sec. Fluorescence data were collected in real time at 1-second intervals for the first 60 seconds and at 6-second intervals for an additional 120 seconds at 23°C. The functional responses were measured and represented as peak fluorescence intensity minus basal intensity, in relative fluorescence units (RFUs) for each concentration of each agonist. 

Data Analysis

The original PI turnover and [Ca²⁺]_i mobilization data were analyzed with the nonlinear, iterative, sigmoidal curve-fitting function of a commercial software program (Origin Scientific Graphics; Microcal Software, Northampton, MA). ^{13 14 17} Agonist potency (EC₅₀) from these assays was defined as the compound concentration eliciting 50% of the maximal functional response induced by the agonist. Antagonist potency (equilibrium drug dissociation constants, K _i) was calculated with the following equation ^{11 17 18} : antagonist K _i = antagonist IC₅₀/[1 + (agonist concentration/agonist EC₅₀)], ^{11 17} where IC₅₀ is the antagonist concentration causing 50% inhibition of the maximal functional response) when the antagonistic effects of multiple concentrations of AL-8810 were titrated against a fixed concentration of the different agonists used in the current studies. [Ca²⁺]_i mobilization fluorescence traces obtained from the FLIPR-based assays were amalgamated by using the graphics software, to show the concentration-dependent nature of the agonist-stimulated responses. 

All data were calculated and represented as the mean ± SEM. A Student’s unpaired t-test was used to determine statistical differences (if any) between the agonist potencies of the various compounds tested. P < 0.05 was set as the minimum acceptable level of significance between data sets. 

Results

PI turnover in the h-TM cells was linear at least up to 90 minutes (data not shown) and thus a 60-minute incubation protocol was chosen for the agonist-induced generation of [³H]-IPs, to maximize the signal-to-noise ratio and to reduce possible hydrolysis or degradation of the test compounds. This assay paradigm was identical with that which has been used to characterize the FP-receptor-induced PI turnover in Swiss 3T3 mouse fibroblasts, ^{13 14} rat vascular smooth muscle cells (A7r5), ^{11 16} and HEK-293 cells expressing the cloned human ciliary body-derived FP receptor. ^{15 19} In some typical experiments involving FP agonists such as (±)-fluprostenol, travoprost, and the free acids of latanoprost and travoprost, the amounts of [³H]-IPs generated in h-TM cells were as follows: basal level, 1925 ± 206 dissociations per minute (dpm; n = 8); maximum stimulation (using 1–10 μM agonists), 7780 ± 1155 dpm. Thus, there was a 4.0 ± 0.36-fold stimulation of PI turnover above the basal level, thereby yielding a more than adequate signal-to-noise ratio. 

The various prostaglandin analogues tested in the current studies stimulated accumulation of [³H]-IPs in h-TM cells in a concentration-dependent manner (Fig. 1) akin to that previously reported for many other cell types expressing native or recombinant FP receptors. ^{11 13 14 15 16} The relative potencies (EC₅₀s) of these and other agonists are shown in Table 1 . These data compared favorably with those reported for the FP receptor in several other cell types. ^{11 13 14 15 16 19} The rank order of potency of the PG free acids tested in h-TM cells was: travoprost acid (EC₅₀ = 2.4 ± 0.7 nM) > cloprostenol (EC₅₀ = 4.5 ± 1.3 nM) > (±)-fluprostenol (EC₅₀ = 10.8 ± 2.1 nM) > latanoprost acid (EC₅₀ = 34.7 ± 2.4 nM) > bimatoprost acid (EC₅₀ = 112 ± 55 nM) > PGF_2α (EC₅₀ = 120 ± 26 nM) ≫ unoprostone (EC₅₀ = 3280 ± 1830 nM) > S-1033 (EC₅₀ = 4570 ± 2280 nM). Many of the synthetic PGs (e.g., travoprost acid, cloprostenol, (±)-fluprostenol) were more potent than the natural prostaglandin ligand (PGF_2α). Similarly, the racemate (±)-fluprostenol was nearly five times weaker than its (+)-enantiomer (travoprost acid; [+]-fluprostenol; Table 1 ). As expected, the prodrug derivatives of the former compounds exhibited lower potencies than their free acids, with the following ranked order of potency observed: travoprost (isopropyl ester; EC₅₀ = 89 ± 20 nM) > latanoprost (isopropyl ester; EC₅₀ = 778 ± 245 nM) > 0.03% bimatoprost ophthalmic solution (Lumigan; Allergan Inc.; EC₅₀ = 1410 ± 397 nM) ≥ bimatoprost (amide; Cayman; EC₅₀ = 6940 ± 1836 nM; Fig. 1 ; Table 1 ). The EC₅₀s for bimatoprost from the two sources were statistically insignificant (P < 0.079). It was noteworthy that (±)-fluprostenol, latanoprost acid, bimatoprost acid, PGF_2α, unoprostone, and S-1033 exhibited a significantly (P < 0.02 to P < 0.001) lower potency than travoprost acid (Table 1) . Similarly, travoprost (isopropyl ester) was significantly (P < 0.05 to P < 0.01) more potent than other prodrugs such as latanoprost and bimatoprost (Lumigan; Table 1 ). 

To study the downstream events after generation of IPs, limited experiments were conducted to study mobilization of [Ca²⁺]_i in the h-TM cells with a few of the FP receptor agonists of different potencies. As shown in Figure 2 , travoprost acid, unoprostone, and S-1033 rapidly (within a few seconds) induced [Ca²⁺]_i mobilization in h-TM cells in a concentration-dependent manner. The [Ca²⁺]_i mobilization was a transient response measured only over a 3-minute period and thus represented a nonequilibrium situation. However, once again, travoprost acid was found to be more potent than the other FP agonists studied, based on their EC₅₀ values: unoprostone, 2400 ± 656 nM; S-1033, 1080 ± 220; PGF_2α, 98.6 ± 26.7 nM; and travoprost acid, 26; 38 nM. Travoprost acid’s potency in the [Ca²⁺]_i mobilization assay compared well with that reported for the mouse FP receptor (EC₅₀ = 47 nM). ¹⁵  

To further characterize the pharmacology of the receptor responding to the aforementioned FP-class prostaglandins, we explored the antagonism of these responses by the FP-receptor-selective antagonist, ¹¹ AL-8810. Here, AL-8810 concentration dependently antagonized the (±)-fluprostenol-induced PI turnover responses in the h-TM cells (K _i = 2.56 ± 0.62 μM; Fig. 3 ). In additional experiments, AL-8810 also antagonized PI turnover induced by bimatoprost (K _i = 1.0 μM), travoprost acid (K _i = 2.5 μM), latanoprost acid (K _i = 4.3 μM), and unoprostone (K _i = 2.4 μM). The FP receptor antagonist potency of AL-8810 at the cloned human ciliary body FP receptor was quite similar (K _i = 1–2 μM; described later). 

The agonist and antagonist potencies of these various prostaglandin analogues at the h-TM cell FP receptor, determined using the PI turnover assays, correlated well with their functional potencies determined at the cloned human ciliary body FP receptor (r = 0.92; P < 0.0001; Fig. 4 ). In addition, the FP-receptor-induced PI turnover data obtained from h-TM cells also were highly correlated with data obtained from other cell types and assays involving FP receptors as follows: h-TM versus mouse 3T3 cells, r = 0.91; h-TM versus rat A7r5 cells, r = 0.94; h-TM versus [³H]-PGF_2α binding, r = 0.9 (data not shown). 

Discussion

The recent report of Anthony et al. ⁹ provided initial evidence for the existence of putative FP receptors stimulating PI turnover in h-TM cells, but only PGF_2α was used to investigate the PI-turnover response. ⁹ However, PGF_2α binds to several different PG receptors and is not very selective ^{1 18} ; hence, the unequivocal identification of functionally active FP receptors in h-TM cells remained to be demonstrated. In the present study, we extended these initial observations using h-TM cells obtained from eyes of a large number of donors and by using several synthetic FP-class PG analogues (free acids, isopropyl esters, and an amide), some of which have been shown to be highly selective for the FP receptor and lower IOP in various mammalian species, including humans. We also demonstrated that many of the FP agonists also rapidly mobilized [Ca²⁺]_i in the h-TM cells. Furthermore, the FP-nature of the h-TM receptor responding to the PG analogues studied was confirmed by the use of the FP-receptor antagonist, AL-8810. ^{11 15 19}  

Specifically, whereas PGF_2α was not a very potent compound in the h-TM PI turnover assay, various synthetic analogues of PGF_2α were highly potent and efficacious agonists, with travoprost acid, cloprostenol, and (±)-fluprostenol exhibiting nanomolar potencies. The free acids of these PG analogues (e.g., travoprost acid, cloprostenol, (±)-fluprostenol, latanoprost acid, bimatoprost acid, and unoprostone) exhibited greater potencies than the respective isopropyl esters and amide prodrug derivatives of these PGs, thus indicating that the free acids represent the active moieties of these IOP-lowering agents. Travoprost acid ((±)-fluprostenol) was approximately five times more active than the racemic (±)-fluprostenol in the PI turnover assays, and it was the most potent agonist (travoprost EC₅₀ = 2.4 nM) in the h-TM cells. This high functional potency of travoprost acid in the h-TM cells was also observed at the cloned human ciliary body FP receptor (EC₅₀ = 3.2 nM) ¹⁹ and the mouse FP receptor (EC₅₀ = 2.7 nM). ¹ The potency of the natural prostaglandin agonist PGF_2α (EC₅₀ = 120 nM) at the h-TM FP receptor, detected by PI turnover, matched that previously reported for h-TM cells (EC₅₀ = 100 nM), ⁹ but unfortunately no other comparative data for other FP agonists in the h-TM cells are available in the literature. However, the relative agonist and antagonist potencies (and the ranked order of potency) of the PG analogues studied in the h-TM cells in our current studies were similar (r = 0.92) to those found for the FP receptor cloned from the human ciliary body. ^{10 19} Furthermore, the relatively high degree of correlation between the potencies of up to eight FP agonists and an antagonist at the h-TM cells and those at the mouse ^{13 14} and rat ¹⁶ FP receptor supported the close homology of the amino acid sequences (and intracellular signaling mechanisms) of the FP receptor in these different species, determined by molecular biological techniques. ²⁰  

It was considered important to study the downstream signal-transduction events related to FP receptor agonist-induced PI turnover. PI hydrolysis is known to result in release of Ca²⁺ from intracellular stores. ^{9 13 15} Accordingly, we observed a rapid and concentration-dependent mobilization of [Ca²⁺]_i in h-TM cells by several FP agonists, including travoprost acid, unoprostone, and S-1033. Even though the [Ca²⁺]_i mobilization assay is conducted under transient and nonequilibrium conditions because of the real-time nature of this technique, with observations being made over seconds, ¹⁵ the potencies of the compounds obtained from the PI turnover assay ([³H]-IP accumulation over 60 minutes) and the latter [Ca²⁺]_i assay were of similar magnitude. More important, travoprost acid was still the most potent and efficacious agonist in the [Ca²⁺]_i mobilization assay in the h-TM cells, akin to the PI-turnover assay findings. 

The recent detection of FP receptor mRNA in human TM biopsy specimens ²¹ and TM cells ⁹ determined by RT-PCR technique supported our current biochemical and pharmacologic functional studies on the h-TM cell FP receptor. Cultured h-TM cells produce PGE₂ and PGF_2α ²² and FP-receptor-mediated generation of PGE₂ has been reported. ²³ Because these endogenous PGs have been shown to regulate aqueous humor dynamics in human eye anterior segments, ^{4 24} functional activation of the h-TM cell FP receptors may therefore play a central role in the PG-mediated regulation of IOP by promoting conventional outflow in addition to the uveoscleral outflow. In addition, because production of matrix metalloproteinases (MMPs) by ciliary muscle cells in response to FP agonists has been implicated as a mechanism of action for latanoprost, ²⁵ it is possible that the FP receptor in h-TM cells has a similar role. However, further work is needed to explore this possibility. 

The prodrug bimatoprost has recently become available to treat ocular hypertension. ⁵ Unoprostone isopropyl ester ^{3 26} is another PG analogue prodrug that has been available for the same indication. Recent reports have suggested that bimatoprost ⁵ and unoprostone isopropyl ester ^{27 28} do not interact with any PG receptors to induce biological responses. However, the present studies in h-TM cells provide strong evidence for the FP receptor agonist nature of both of these compounds and their respective free acids in both the PI turnover assay and [Ca²⁺]_i mobilization assays. These data in h-TM cells have further substantiated and underscored other recent findings of FP agonist effects of bimatoprost and its free acid (and also of unoprostone; UF-021) at the cloned human ciliary body FP receptor. ^{15 19 29} Bimatoprost free acid (17-phenyl-trinor PGF_2α) has also been shown to be an agonist at the constitutive mouse ^{13 14} and rat ¹⁶ FP receptor and the recombinant human ocular FP receptor, ^{19 29} in PI turnover assays. Similarly, unoprostone (UF-021; 13,14-dihydro-15-keto-20-ethyl-PGF_2α) competes for [³H]-PGF_2α binding to bovine corpus luteum FP receptors and also behaves as an FP receptor agonist at the mouse ¹⁴ and human ¹⁹ FP receptor, stimulating PI turnover and mobilizing intracellular Ca²⁺. Therefore, both bimatoprost and unoprostone bind to and activate FP prostaglandin receptors to induce second-messenger functional responses. 

Travoprost acid ([+]-fluprostenol) exhibited a greater overall efficacy than bimatoprost and unoprostone in the h-TM cell PI turnover assays (Fig. 1) with general confirmation in the h-TM cell [Ca²⁺]_i mobilization assay, in this study and elsewhere. ¹⁵ Similar high potency and efficacy observations for travoprost acid have also been made in other cell types. ^{14 15 19} Although the reasons for the higher potency and efficacy of travoprost acid (relative to unoprostone (acid) and bimatoprost (amide)) are not fully understood, they may be related to its high FP receptor selectivity ^{1 7} and also may be because it is the more potent (+)-enantiomer of racemic fluprostenol. These attributes of travoprost acid may permit the formation of a more stable and better-coupled ligand-receptor complex that favors a greater and faster recruitment of the appropriate G proteins and phospholipase C, thus resulting in an overall greater activation of the signal-transduction processes associated with the FP-receptor. However, additional work is needed to explore these possibilities further. 

In conclusion, we determined the pharmacologic properties of the endogenously expressed FP receptor present in h-TM cells of several human donors, by using numerous synthetic prostaglandin FP receptor agonist analogues and an FP-receptor-selective antagonist. In addition, we demonstrated that bimatoprost, unoprostone isopropyl ester, and their respective free acids are agonists at the h-TM FP receptor where they readily induce the generation of [³H]-IPs and rapidly and directly mobilize [Ca²⁺]_i through the FP prostaglandin receptor. 

 Submitted for publication April 2, 2002; revised July 29, 2002; accepted August 19, 2002.

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Figure 1.

 

Concentration-dependent stimulation of [³H]-IP formation in h-TM cells induced by various FP receptor PG agonist analogues. The h-TM cell membranes were radiolabeled with [³H]-myo-inositol after a 24- to 30-hour incubation. After cells were rinsed, different concentrations of the agonists were added in culture medium containing 10 mM LiCl for 1 hour at 37°C. The assays were terminated, and anion-exchange chromatography and liquid scintillation spectrometry were used to isolate and quantify, respectively, the total [³H]-IPs. The data were analyzed using a non-linear (sigmoidal-fit), iterative, curve-fitting computer program to obtain the agonist potency values (EC₅₀s) for each agonist (Table 1) and various concentration-response curves constructed. The data are the mean ± SEM of results in more than three experiments, using normal h-TM cells isolated from TM tissues from up to six different human donors without ocular disease history.

Table 1.

 

Agonist Potencies of FP-Class PG Analogues in h-TM Cells, Determined in PI-Turnover Assays

Compound	Functional Potency (EC50, nM)	Statistical Significance
Travoprost acid ([+]-fluprostenol)	2.4 ± 0.7
Cloprostenol (acid)	4.5 ± 1.3	NS*
(±)-Fluprostenol (acid)	10.8 ± 2.1	P < 0.02, †
Latanoprost acid (PHXA85)	34.7 ± 2.4	P < 0.001, †
Bimatoprost acid (17-phenyl-trinor PGF2α)	112 ± 55	P < 0.001, †
Travoprost (isopropyl ester)	89 ± 20
PGF2α (acid)	120 ± 26	P < 0.001, ‡
Latanoprost (isopropyl ester)	778 ± 245	P < 0.05, §
Bimatoprost ophthlamic solution; amide (Lumigan)	1410 ± 379	P < 0.05, §
Unoprostone isopropyl ester	2310 ± 1240	NS, ∥
Unoprostone (acid; UF-021)	3280 ± 1830	P < 0.001, ‡
Bimatoprost (amide)	6940 ± 1830	P < 0.01, §
S-1033 (acid)	4570 ± 2280	P < 0.05, ‡

Figure 2.

 

[Ca²⁺]_i mobilization in h-TM cells in response to different FP receptor agonists. The h-TM cells were loaded with the Ca²⁺-sensitive dye for 1 hour at 23°C and then exposed to different concentrations of the various FP receptor agonists on the FLIPR. Real-time [Ca²⁺]_i mobilization in the cells was monitored and recorded over 180 seconds at 23°C and the data analyzed on computer to obtain agonist potencies. Note the rapid concentration-dependent mobilization of Ca²⁺ in these cells by travoprost acid (A), unoprostone (B) and S-1033 (C). Similar data were obtained from other human donor TM cells and the cumulative agonist potencies calculated (see Results section).

Figure 3.

 

Concentration-dependent inhibition of (±)-fluprostenol-induced formation of [³H]-IPs formation by AL-8810 in normal h-TM cells isolated from numerous human donors. The h-TM cell membranes were radiolabeled with [³H]-myo-inositol after a 24-hour incubation. After rinsing the cells, different concentrations of the FP receptor antagonist, AL-8810, were added to the cells in the culture medium containing 10 mM LiCl and the incubation continued for 15 to 20 minutes at 37°C. After this time, the agonist, (±)-fluprostenol (100 nM final), was added to the cells and the incubation continued for another hour at 37°C. The assays were terminated and anion-exchange chromatography and liquid scintillation spectrometry were used to isolate and quantify, respectively, the total [³H]-IPs. The data were analyzed using a non-linear (sigmoidal-fit), iterative, curve-fitting computer program to obtain the antagonist potency value for AL-8810 and the concentration-response curves constructed as shown. Data are the mean ± SEM of results from more than three experiments. Similar data were obtained for the antagonism by AL-8810 of the PI turnover induced by bimatoprost, unoprostone, and the other FP-receptor PG agonist analogues described in the Results section.

Figure 4.

 

Correlation of the agonist and antagonist potencies of numerous prostaglandin analogues of the FP-class at the FP receptors in normal h-TM cells and at the cloned human ciliary body derived FP receptor. Agonist potencies of various FP analogues (and antagonist potencies of AL-8810) were determined in normal primary h-TM cells from several human donors and in HEK-293 cells expressing the cloned human ciliary body FP receptor ^{15 19} using the PI turnover assay. The −log of the EC₅₀ (pEC₅₀) and -log of the K_i (pK_i) for the 14 prostaglandins (13 agonists and 1 antagonist) were calculated to construct the correlation plot. Note the high degree of correlation between the two different FP receptor systems (r = 0.92; P < 0.0001). High correlations were also obtained between the h-TM cells and other cells derived from mouse and rat tissues expressing endogenous FP receptors (see Results section).

The authors thank Mari Engler for the initial cultures of h-TM cells, colleagues in the Medicinal Chemistry Unit for synthesis of certain PG analogues used in the current studies, and Tom Dean and Mark Hellberg for helpful comments on the manuscript.