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From the Molecular Pharmacology Unit, Glaucoma Research, Alcon Research, Ltd., Fort Worth, Texas.
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Abstract |
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(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) > PGF2 (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, PGF2, 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 (Ki = 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.
ethyl amide),5 has also recently been marketed for this indication. Even though putative FP receptors have been detected in the human ciliary muscle6 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 11ß-fluoro-15-epi-15-indanyl-PGF2 (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 |
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Cell Culture
The h-TM cells were obtained as previously described12 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, Ki) was calculated with the following equation11 17 18 : antagonist Ki = 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.
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Results |
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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) > PGF2 (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 (PGF2). 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, PGF2, 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 ).
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, 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
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binding, r = 0.9 (data not shown).
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Discussion |
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was used to investigate the PI-turnover response.9 However, PGF2 binds to several different PG receptors and is not very selective1 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 PGF2 was not a very potent compound in the h-TM PI turnover assay, various synthetic analogues of PGF2 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 PGF2 (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 mouse13 14 and rat16 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 specimens21 and TM cells9 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 PGF222 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 ester3 26 is another PG analogue prodrug that has been available for the same indication. Recent reports have suggested that bimatoprost5 and unoprostone isopropyl ester27 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 PGF2) has also been shown to be an agonist at the constitutive mouse13 14 and rat16 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-PGF2) competes for [3H]-PGF2 binding to bovine corpus luteum FP receptors and also behaves as an FP receptor agonist at the mouse14 and human19 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 selectivity1 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.
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Acknowledgements |
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Footnotes |
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Commercial relationships policy: E, F.
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: Najam A. Sharif, Director and Head, Molecular Pharmacology Unit, Alcon Research, Ltd. (R2-19), 6201 South Freeway, Fort Worth, TX 76134-2099; naj.sharif{at}alconlabs.com.
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References |
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analogue Adv Prostaglandin Thrombox Leukotr Res 23,513-518
receptors in the human trabecular meshwork Invest Ophthalmol Vis Sci 39,315-321(PGF2) receptor J Biol Chem 272,27147-27154 analog with selective antagonist effects at the prostaglandin F2 (FP) receptor J Pharmacol Exp Ther 290,1278-1284 binding to EP3, and FP prostaglandin receptor binding sites in bovine corpus luteum: characterization and correlation with functional data J Pharmacol Exp Ther 286,1094-1102 and its analogs induce release of endogenous prostaglandins in iris and ciliary muscles isolated from cat and other mammalian species Exp Eye Res 63,305-310[CrossRef][Medline][Order article via Infotrieve]
by human and rabbit ocular tissues and agonist activity of bimatoprost and 17-phenyl-trinor PGF2 ARVO Abstract #4080This article has been cited by other articles:
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  C B Camras, N A Sharif, M B Wax, and J Stjernschantz Bimatoprost, the prodrug of a prostaglandin analogue Br. J. Ophthalmol., June 1, 2008; 92(6): 862 - 863. [Full Text] [PDF]
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