Introduction

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The classification of prostanoid [prostaglandin (PG)] receptors according to the binding affinities, potencies, and selectivities of the five known classes of endogenous PGs (PGD₂, PGE₂, PGF_2a, PGI₂, and thromboxane A₂), as first proposed by Coleman et al. (1990), has been validated in numerous studies by using pharmacological methods and molecular biological techniques (Coleman et al., 1994; Narumiya, 1994). All PG receptors identified to date are seven-transmembrane proteins that couple to specific G proteins mediating the formation of cAMP or inositol trisphosphate/diacylglycerol second messengers (Coleman et al., 1994). Isoforms of some PG receptors, e.g., the EP, TP, and prostaglandin PGF₂ (FP) receptors, have been positively identified, although full characterization of the pharmacological properties of all such isoforms generally has not yet been accomplished (Coleman et al., 1994; Narumiya, 1994; Sugimoto et al., 1994; Pierce et al., 1997). Potent, selective, synthetic agonists of some PG receptors have defined the specific functional responses coupled to activation of those receptors by use of cell lines expressing the respective cloned or endogenous PG receptors and also isolated tissues (Coleman et al., 1994). However, conclusive identification of the specific receptor(s) involved in PG-mediated functional responses of complex biological systems in vitro or in vivo requires potent, selective receptor antagonists. To date, of the eight known major PG receptor subtypes, experimentally useful antagonists, e.g., possessing both the requisite potency and selectivity, have been described only for the DP receptor (BW A868C) (Giles et al., 1989) and TP receptor (SQ-29,548) (Ogletree et al., 1985), whereas less robust antagonists with modest to poor selectivity have been characterized for the EP₁ receptor (SC51089 and AH6809, which is also a weak antagonist at the EP₂ and DP receptors) and EP₄ receptor (AH23848) (Coleman et al., 1994).

FP receptor agonists are potent, highly efficacious agents that reduce elevated intraocular pressure in humans and certain animals (Wang et al., 1990; Bito, 1997) and that have other pharmacological effects in the mammalian body (Coleman et al., 1994). The major FP receptor isoform has high-sequence homology among many animal species (Abramovitz et al., 1994, 1995; Lake et al., 1994; Sakamoto et al., 1994); a second, minor FP receptor isoform with a truncated carboxyl terminus has been detected thus far only in a highly specialized cell type (large cell of mid-cycle corpus luteum) of sheep (Pierce et al., 1997). The structure, signaling properties, and pharmacological profile of the single FP receptor in humans and several species have been investigated. Agonist binding to the FP receptor activates phospholipase C (PLC), producing elevated diacylglycerol and inositol trisphosphate and a rapid increase in intracellular Ca²⁺ as early-signaling events (Davis et al., 1987; Woodward et al., 1990; Sakamoto et al., 1994; Pierce et al., 1997; Griffin et al., 1997, 1998). Numerous such studies have demonstrated considerable similarity of FP receptor function across species, corroborating predictions based on the high interspecies homology of the FP receptor gene. However, the tissue distribution of the FP receptor in ocular as well as nonocular tissues varies greatly among species (Bhattacherjee and Paterson, 1994; Coleman et al., 1994; Lake et al., 1994; Narumiya, 1994; Abramovitz et al., 1995; Ocklind et al., 1997; Mukhopadhyay et al., 1999; Davis and Sharif, 1999), raising important questions about its fundamental physiologic functions. Despite substantial efforts in this area, there is still no well characterized FP receptor antagonist sufficiently potent and selective to address these issues. A few structural analogs of PGF₂, such as PGF₂ dimethylamine and PGF₂ dimethylamide (Stinger et al., 1982) and some nonprostanoid structures, i.e., phloretin (Kitanaka et al., 1993) and glibenclamide (Delaey and Van de Voorde, 1995), have been reported to be FP receptor antagonists. However, the potencies and selectivities of these compounds at the FP receptor relative to other prostanoid receptors are not well characterized.

This report describes in vitro studies with AL-8810 [(5Z, 13E)-(9S,11S,15R)-9,15-dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20-pentanor-5,13-prostadienoic acid] (Fig. 1), a novel, low-efficacy FP receptor agonist with selective antagonistic activity at the FP receptor. We reported recently the pharmacological properties of the FP receptor of Swiss mouse 3T3 fibroblasts (Griffin et al., 1997) and A7r5 rat thoracic aorta vascular smooth muscle cells (Griffin et al., 1998). The large amplification of FP receptor-coupled formation of inositol phosphates (IPs) by both cell types made this response the pharmacological method of choice for characterizing the activities of AL-8810 at the FP receptor in this study. These results suggest the potential utility of AL-8810 as an FP receptor antagonist for elucidating FP receptor involvement in PG-initiated responses of complex biological systems containing heterogeneous PG receptor populations.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structure of AL-8810.

	Materials and Methods

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Cell Culture. A7r5 rat vascular smooth muscle cells and Swiss mouse 3T3 fibroblasts were maintained and cultured by standard procedures, as described previously (Griffin et al., 1998). The culture medium was Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/liter glucose and 110 mg/liter sodium pyruvate, supplemented with 2 mM L-glutamine, 10 µg/ml gentamicin sulfate, and 10% fetal bovine serum. Other cells used in these studies included: embryonic bovine tracheal (EbTr) cells (Ito et al., 1990; Crider et al., 1999), transformed human nonpigmented ciliary epithelial (NPE) cells (Jumblatt et al., 1994; Crider et al., 1998a), both grown in the above culture medium; Chinese hamster ovary (CHO-K1) cells (Milne et al., 1994), grown in Ham's F-12 medium with other supplements as listed above; and NCB-20 cells, cultured in the DMEM-based medium as described above, containing also 2% hypoxanthine, aminopterin, and thymidine (Blair et al., 1980; Crider et al., 1998b). Cells were maintained in a humidified atmosphere of 5% CO₂ and 95% air, with two changes of fresh medium weekly. All cells were passaged at approximately 90% confluence by treatment with 0.05% trypsin/0.53 mM EDTA.

Second Messenger Assays. Previously published procedures were used to measure [³H]inositol phosphates ([³H]IPs) produced by agonist-mediated activation of PLC (Sharif et al., 1994, 1998; Griffin et al., 1997, 1998). In brief, cells grown to confluence in 24-well, uncoated plastic plates were exposed for 24 to 30 h to 1.0 to 1.5 µCi [³H]myo-inositol (18.3 Ci/mmol, as supplied) in 0.5 ml of DMEM (serum-free, containing unlabeled myo-inositol), corresponding to a specific activity of 37.5 µCi/mmol [³H]myo-inositol in the labeling medium. Cells then were rinsed once with DMEM/F-12 containing 10 mM LiCl and incubated with agonist in the same medium for 1 h at 37°C (triplicate determinations for each concentration). Antagonist effects were determined by adding the antagonist (or the solvent ethanol as a control) for 10-20 min (as described in the figure legends) before the 1-h incubation with agonist. After aspirating the medium, cells were lysed with 1 ml of cold (4°C) 0.1 M formic acid. The chromatographic separation of radiolabeled components on an AG-1-X8 column was performed exactly as described (Griffin et al., 1997, 1998). The total [³H]IPs eluted with 4 ml of 1.2 M ammonium formate (containing 0.1 M formic acid) was mixed with 15 ml of scintillation fluid and counted with a beta counter. Detailed procedures for measurement of PG receptor-coupled cAMP formation by an automated radioimmunoassay (RIA) procedure were described recently (Crider et al., 1998b). Confluent cells (grown in 48-well, uncoated plastic plates) were rinsed with DMEM/F-12 and then incubated 20 min with 0.8 mM ascorbate, 1 mM 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), and antagonist, if present in the experiment, before adding the agonist. The medium was aspirated at the end of the agonist stimulation period, and the cells were lysed with 0.1 M acetic acid (4°C), followed by neutralization with 0.1 M sodium acetate (4°C). The cAMP assay was automated by utilizing a Biomek 1000 robot (Beckman Instruments, Fullerton, CA) to dilute samples with RIA buffer into a 96-well filtration plate (0.45-µm surfactant-free mixed cellulose). ¹²⁵I-cAMP and primary cAMP antibody then were added by the robot, which also mixed the samples thoroughly. After an incubation at 4°C for 16 to 24 h, the secondary antibody was added to the samples. After a 20-min incubation at room temperature, bound and free ¹²⁵I-cAMP were separated by vacuum filtration, using a Millipore disposable punch-tip assembly and manifold. The bound ¹²⁵I-cAMP was quantitated by use of a gamma counter and comparison to a standard curve of known cAMP samples carried through the assay procedure.

Materials. Cell lines were purchased from the American Type Culture Collection (Manassas, VA), except for nonpigmented ciliary epithelial (NPE) cells, generously provided by Dr. M. Coca-Prados, and NCB-20 cells, a gift from Dr. M. W. Nirenberg. Life Technologies (Grand Island, NY) supplied DMEM, DMEM/F-12, Ham's F-10, glutamine, gentamicin, and trypsin/EDTA. Fetal bovine serum (HyClone, Logan, UT) was heat-inactivated at 56°C for 30 min and stored at 20°C. Amersham (Deerfield, IL) provided [³H]myo-inositol. Arg⁸-vasopressin (AVP) and [d(CH₂)₅, Tyr(Me)₂, Tyr(NH₂)⁹]-AVP were purchased from Peninsula Laboratories (Belmont, CA). AG 1-X8 anion-exchange resin was a product of Bio-Rad (Hercules, CA). Ecolume scintillation fluid was supplied by ICN Biomedicals (Costa Mesa, CA). The ¹²⁵I-cAMP RIA kits were supplied by PerSeptive Diagnostics (Cambridge, MA).

Data Analyses. Concentration-response data were analyzed using the sigmoidal-fit function of the Origin Scientific Graphics software (Microcal Software, Northampton, MA) to determine agonist potency (EC₅₀ value) and efficacy, relative to a standard full agonist. For the FP receptor, cloprostenol served as the reference standard full agonist. Equilibrium inhibition constants were calculated as K_i = IC₅₀/[(1 + (agonist concentration/agonist EC₅₀)] (Cheng and Prusoff, 1973) when the antagonistic effects of multiple concentrations of AL-8810 were titrated against a fixed concentration (100 nM) of fluprostenol. The apparent pK_b values (log antagonist dissociation constant) were calculated as K_b = (antagonist concentration)/[(agonist EC₅₀ in presence of antagonist/agonist EC₅₀ in absence of antagonist)  1], according to Furchgott (1972). Antagonist potency was represented as pA₂ (log drug dissociation constant) and is defined as log molar antagonist concentration required to shift the agonist concentration-response curve to the right by 2-fold, as determined by Schild analysis (Arunlakshana and Schild, 1959).

	Results

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The concentration-response data shown in Fig. 2 demonstrate that AL-8810 is a weak partial agonist with low efficacy at the FP receptor of A7r5 cells. The mean potency of AL-8810 was EC₅₀ = 261 ± 44 nM (n = 3) and E_max = 19% compared with cloprostenol (EC₅₀ = 0.84 nM; E_max = 100%) (Griffin et al., 1998), the standard full agonist employed routinely in these experiments. The response to 100 nM fluprostenol, a highly selective full agonist at the FP receptor (Coleman et al., 1990; Sharif et al., 1998, 1999), with EC₅₀ = 4.45 nM in this assay (Griffin et al., 1998), was inhibited by a short preincubation (10 min) of the cells with increasing concentrations of AL-8810 (Fig. 3). The mean antagonist potency (K_i) determined from the individual experiments of Fig. 3 (based on Cheng and Prusoff, 1973) was 426 ± 63 nM (n = 5), with pK_i = 6.39 ± 0.06. AL-8810 produced a concentration-dependent dextral shift in the fluprostenol concentration-response curve, without significantly decreasing the agonist-induced maximal response (Fig. 4), typical of a competitive antagonist. Schild analyses of multiple experiments of the type in Fig. 4 (Fig. 5) determined a line with a slope of 0.80 ± 0.08 (different from 1.0), correlation coefficient of 0.96 ± 0.01, and mean pA₂ value of 6.68 ± 0.23 (n = 4), corresponding to a mean antagonist dissociation constant of 285 ± 97 nM (n = 4). As evidence that the protein target of AL-8810 was the FP receptor, and not some other protein early in the signaling pathway, AL-8810 was evaluated for possible antagonist activity at the PLC-coupled V₁-vasopressin receptor of A7r5 cells, using AVP as the agonist. As shown in Fig. 6, AL-8810 produced no measurable inhibition of this response. In the positive control experiment (Fig. 6), the [³H]IPs' response to AVP was completely inhibited by a known selective antagonist of this receptor, [d(CH₂)₅, Tyr(Me)₂, Tyr(NH₂)⁹]-Arg⁸-vasopressin (Thibonnier et al., 1991). In identical experimental protocols (Griffin et al., 1997), the agonist potency and efficacy of AL-8810 at the FP receptor of Swiss mouse 3T3 fibroblasts were determined to be 186 ± 63 nM (n = 3) and 23%, respectively (data not shown). The pA₂ value of AL-8810 with this cell line (Schild slope = 0.92, equal to 1, p < .05) was 6.39 ± 0.09, corresponding to an antagonist dissociation constant of 474 nM (data not shown). We also have observed that AL-8810 inhibits, with similar antagonist potency, fluprostenol-stimulated activation of PLC in HEK-293 cells expressing the cloned human ocular FP receptor (unpublished observations).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for stimulation of PI turnover in A7r5 cells by cloprostenol () and AL-8810 (). Data points are mean values ± S.E. (n = 3) for a representative experiment of three performed.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Inhibition of fluprostenol-stimulated PI turnover response of A7r5 cells by AL-8810. Cells were incubated with AL-8810 for 10 min before adding 100 nM fluprostenol. The mean normalized responses (±S.E.M.) for five independent experiments are shown, with n = 3 replicates in each experiment.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of varying AL-8810 concentration on the concentration-response curves of fluprostenol. Vehicle () or AL-8810 (, 106 M; , 105 M; , 3 × 105 M) was added 10 min before adding various concentrations of fluprostenol. Mean responses ± S.E. (n = 3) are shown for a representative experiment of four experiments performed. In the absence of AL-8810, the EC50 of fluprostenol in this experiment was 3.59 nM.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Schild analysis of antagonism by AL-8810 of fluprostenol-induced PI turnover response in A7r5 cells. Data (n = 13) were generated in four experiments of the type shown in Fig. 4. Linear regression analysis yielded a slope of 0.80 ± 0.08 and correlation coefficient of 0.96 ± 0.01.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of AL-8810 and a selective V1-vasopressin receptor antagonist on the PI turnover response of A7r5 cells stimulated by 100 nM AVP. Cells were preincubated for 15 min with AL-8810 () or 30 min with [d(CH2)5, Tyr(Me)2, Tyr(NH2)9]-Arg8-vasopressin () before stimulation with AVP for 1 h. The response is the mean ± S.E. (n = 3) normalized by the control response (solvent substituted for the antagonist) for one representative experiment of three performed.

A series of experiments was performed to determine the PG receptor selectivity of AL-8810 by assessing its antagonist activity at other PG receptors, which are known to be expressed endogenously in certain established cell lines. In the concentration range of 1 nM to 100 µM, only 100 µM AL-8810 produced any significant inhibition of TP receptor-mediated activation of PLC in human NPE cells (Fig. 7). By comparison, this response was completely inhibited by the potent, selective TP receptor antagonist SQ-29,548, with IC₅₀ = 0.61 µM (Fig. 7). The NPE cell TP receptor is activated selectively by two known TP receptor ligands, I-BOP (EC₅₀ = 8.2 nM) and U-46619 (EC₅₀ = 1.23 µM) and has been identified as the TP isoform (unpublished observations). As shown in Fig. 8, AL-8810 was evaluated for antagonist activity at several adenylyl cyclase-coupled PG receptors: after a short preincubation with increasing concentrations of AL-8810, each receptor was activated by its respective endogenous ligand. In these experiments, AL-8810 produced no statistically significant inhibition of cAMP formation coupled to the following PG receptors: 1) EP₂ receptor of human NPE cells (Jumblatt et al., 1994; Crider et al., 1998a); 2) EP₄ receptor on CHO cells (Milne et al., 1994); 3) DP receptor of EbTr cells (Ito et al., 1990; Crider et al., 1999); and 4) IP receptor on NCB-20 cells (Blair et al., 1980; Crider et al., 1998b). Data published by our laboratory have confirmed the identity of these PG receptors, as follows: 1) EP₂ receptor, activated by the selective EP₂ receptor agonist butaprost (EC₅₀ = 212 nM) and other known ligands of the EP₂ receptor (Crider et al., 1998b); 2) EP₄ receptor, not activated by butaprost but inhibited by the EP₄-selective antagonist AH23848B (J.Y.C., B.W.G., and N.A.S., in preparation); 3) DP receptor, high potencies of selective DP receptor ligands [ZK118182 (16 nM), RS-93520 (23 nM), BW245C (59 nM), and PGD₂ (100 nM)] and also potent inhibition of the response to these agonists by the selective DP receptor antagonist BWA868C (pA₂ = 8.00) (Crider et al., 1999); and 4) IP receptor, selective and potent activation by PGI₂ (3.5 nM), carbaprostcyclin (5.1 nM), and iloprost (59 nM) (Crider et al., 1998b).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects of AL-8810 and SQ-29,548 on the PI turnover response of NPE cells stimulated by 10 µM U-46619. Cells were preincubated for 15 min with AL-8810 () or SQ-29,548 () before stimulation with 10 µM U-46619 for 1 h. The response is the mean ± S.E. (n = 3) normalized by the control response (solvent substituted for the antagonist) for one representative experiment of three performed. The basal response corresponded to 982 dpm, and the control response (ethanol preincubation followed by 10 µM U-46619) corresponded to 2038 dpm.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of AL-8810 on the functional responses of various adenylyl cyclase-coupled PG receptors. In all experiments, AL-8810 was preincubated for 15 min with the cells, followed by stimulation with the stated prostaglandin, at the concentration and for the time indicated; control cAMP response with the agonist and solvent substituted for the antagonist is given in parentheses for each PG receptor type. , EP2 receptor of NPE cells; 1 µM PGE2 for 60 min (48 pmol of cAMP); , EP4 receptor of CHO-K1 cells; 300 nM PGE2 for 5 min (8.8 pmol of cAMP); , DP receptor of EbTr cells; 1 µM PGD2 for 60 min (23 pmol of cAMP); , IP receptor of NCB-20 cells; 10 nM PGI2 for 15 min (102 pmol of cAMP). In each system, the response in the presence of AL-8810 was normalized by the control response (ethanol vehicle substituted for AL-8810).

We attempted without success to confirm reports (Stinger et al., 1982) that PGF₂ dimethylamine and PGF₂ dimethylamide are antagonists at the A7r5 cell FP receptor. Only the amide analog displayed any measurable activity that was agonistic in nature, producing (at 10 µM concentration) a response <25% of the maximal fluprostenol-stimulated IP response. Significantly, neither of these PGF₂ analogs had any demonstrable antagonistic activity at the FP receptor of A7r5 cells or Swiss mouse 3T3 fibroblasts (data not shown). In preliminary experiments we also have evaluated the antagonist activities of phloretin and glibenclamide at the A7r5 cell FP receptor. The results showed convincingly that both compounds are less potent FP receptor antagonists than AL-8810: the pK_b value of phloretin is less than 5.5 and that of glibenclamide is less than 4.0 (unpublished data). Based on these findings, AL-8810 appears to be the most potent FP receptor antagonist of any compound that has been claimed in the literature to have antagonistic activity at the FP receptor.

	Discussion

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In these studies, we have identified AL-8810 as a unique partial agonist with approximately 20% efficacy at the endogenous FP receptors in cell lines of two different species, Swiss mouse 3T3 fibroblasts and A7r5 rat vascular smooth muscle cells. The FP receptor antagonist activity suggested by these pharmacological properties was confirmed; thus, AL-8810 was shown to be an antagonist of the PI turnover signal-transduction mechanism coupled to FP receptors in both cell types of our study. Moreover, AL-8810 exhibited no significant antagonistic activity at several adenylyl cyclase-coupled PG receptors, including the DP, EP₂, EP₄, and IP receptors, using well characterized and pharmacologically validated assays in our laboratory. For example, the DP receptor of EbTr cells has been shown to be selectively activated by PGD₂ and by structurally similar PGD₂ analogs, and the cAMP response to these ligands is antagonized by the selective DP receptor antagonist BWA 868C (Crider et al., 1999) with the expected high potency (Giles et al., 1989). Similarly, the EP₄ receptor on CHO-K1 cells has been shown to be pharmacologically distinct from the well characterized EP₂ receptor on NPE cells (Jumblatt et al., 1994; Milne et al., 1994; Crider et al., 1998b), as established by the use of the EP₂-selective agonist butaprost and the EP₄-selective antagonist AH23848B (unpublished observations). Moreover, stimulation of the PLC-coupled V₁-vasopressin receptor in A7r5 cells by AVP, a response that was fully inhibited by the potent, selective AVP antagonist [d(CH₂)₅, Tyr(Me)₂, Tyr(NH₂)⁹]-Arg⁸-vasopressin (Thibonnier et al., 1991), was unaffected by high concentrations of AL-8810. Because AL-8810 is a PG-like lipophilic molecule, the small, apparent inhibition of TP receptor function produced by the highest concentration (100 µM) of AL-8810 may result from interactions of the compound with lipid molecules in the cell membrane or with hydrophobic regions of the TP receptor itself that indirectly perturb TP receptor function. However, such generalized "antagonistic" effects observed only at very high concentrations of lipophilic molecules are difficult to characterize and classify. Collectively, these data suggest that AL-8810 exerts a rather selective antagonistic function at the FP receptor.

The activities of AL-8810 as a weak partial agonist and an antagonist at the FP receptor of Swiss mouse 3T3 fibroblasts were very similar to its respective activities at the rat A7r5 cell FP receptor. The antagonistic effects of AL-8810 at the FP receptor appeared to exhibit features of competitive antagonism: for one cell type, the Schild slope was not different from 1, and for both cell types, the antagonist potency/dissociation constants computed by several methods were generally in good agreement. The partial-agonist properties of AL-8810 and cell-specific characteristics, such as receptor density, etc., may contribute, in part, to some differences between the two cell types. The Schild plot slope also could be influenced by AL-8810 perturbation of the cell membrane at high antagonist concentrations, in a manner suggested above for the TP receptor. Studies to address these issues are now in progress. The similar antagonistic properties of AL-8810 at the FP receptors of two different species, along with the evidence for FP receptor selectivity of AL-8810, support other published data indicating very similar pharmacological and structural properties of the FP receptors of these and other species (Abramovitz et al., 1994; Griffin et al., 1997, 1998; Sharif et al., 1998). We also have determined (unpublished data) that AL-8810 has similar antagonistic potency (pA₂ ~6.0) at the cloned human ocular FP receptor expressed by HEK-293 cells. These results on AL-8810 pharmacological activities at various FP receptors reinforce published data that indicate a high degree of interspecies homology of the FP receptor. Moreover, these data suggest that AL-8810 may be a useful experimental tool for investigating FP receptor-mediated functional responses in complex biological systems of different species.

There are several reported FP receptor antagonists in the scientific literature, including two PGF₂ structural analogs, PGF₂ dimethylamine and PGF₂ dimethylamide (Stinger et al., 1982), as well as phloretin (Kitanaka et al., 1993) and glibenclamide (Delaey and Van de Voorde, 1995), that are not prostanoids. In identical experimental protocols conducted in parallel with the experiments with AL-8810, we detected no antagonistic activity of PGF₂ dimethylamine or PGF₂ dimethylamide (unpublished observations). We recently have performed similar experiments with phloretin and glibenclamide and shown these compounds to be quite weak antagonists not only at the FP receptor but also at several other PG receptors (data not shown), confirming previous reports (Kitanaka et al., 1993; Delaey and Van de Voorde, 1995). The data reported in this study were obtained under carefully controlled and very similar experimental conditions using well established functional assays for various PG receptors. This approach allows a more valid comparison of the activities of AL-8810 and other tested antagonists at each of the PG receptors. As such, these results provide definitive evidence for the FP receptor potency and selectivity of AL-8810, a compound with certain structural features in common with PGF₂, the endogenous FP receptor ligand. Additional studies are in progress to demonstrate further the utility and advantages of AL-8810 over other claimed FP receptor antagonists.

In summary, AL-8810, a novel structural analog of PGF₂, is a partial agonist with low efficacy and also a competitive antagonist at the endogenous FP receptors in two cell lines derived from different species. The potency and selectivity of AL-8810 at the FP receptor established by these studies suggest that AL-8810 may be a valuable probe for elucidating specific FP receptor-mediated functions in complex biological systems.