Introduction

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The physiological and pharmacological effects of endogenous and synthetic prostanoids have been characterized in many in vitro and in vivo models and in more limited studies in humans (Coleman et al., 1981, 1994; Beckmann et al., 1988; Mitchell et al., 1994). These studies have established that PGs produce diverse and complex physiological effects, which are mediated by membrane-bound receptors that exhibit some degree of selectivity for the natural PGs, namely PGD₂, PGE₂, PGF₂, PGI₂ (prostacyclin) and thromboxane A₂ (Coleman et al., 1990, 1994; DiMarzo, 1995). The current nomenclature for PG receptors (Coleman et al., 1994) defines the following receptor subtypes, which are believed to exist in the mammalian body: DP, EP (with further subtypes EP₁, EP₂, EP₃ and EP₄), FP, IP and TP (table 1). Due to alternative splicing at the genomic level, further subtypes of the EP₃ receptor have been proposed (Coleman et al., 1994). The major determinants of this nomenclature have been the relatively recent availability of some selective agonists and, to a lesser degree, a few selective antagonists. The genes coding for all major classes of known PG receptors, from animal and human tissues, have now been cloned and expressed in various host cells (Narumiya, 1994; Coleman et al., 1994; Abramovitz et al., 1994; Boie et al., 1995) and the resultant receptor proteins biochemically and pharmacologically verified. These data, together with amino acid sequence data, have confirmed that all of the PG receptor proteins belong to the superfamily of G-protein-coupled receptors having seven transmembrane domains (Narumiya, 1994). The degree of homology among PG receptors of different classes can range up to 40% for the full-length sequence (approximately 400 amino acids) but is generally higher among the transmembrane domains of closely related proteins of this superfamily (Narumiya, 1994; Lake et al., 1994; Boie et al., 1995).

View this table: [in this window] [in a new window]   TABLE 1 Current pharmacological classification of major PG receptor subtypes The table shows the relative selectivity of specific agonist and antagonist PGs for the different PG receptor subtypes based on the work of Coleman et al. (1994).

With regard to coupling of these PG receptors to distinct G-proteins and enzymes mediating the production of intracellular second messengers, the following information is available: FP, TP and EP₁ receptors belong to one subfamily of PG receptors that couple to G_q or G_q/11, resulting in formation of inositol trisphosphate and mobilization of intracellular Ca⁺⁺ (Narumiya, 1994; Coleman et al., 1994); the DP and IP receptors couple to G_s, with consequent activation of adenylyl cyclase and production of cAMP (Sugama et al., 1989; Namba et al., 1994; Boie et al., 1995). At present, several subtypes of EP receptors have been identified, which couple to various G-proteins (Narumiya, 1994; Negishi et al., 1995). Molecular biology techniques have revealed that the EP₃ receptor subtypes are highly homologous except in regions of the cytoplasmic peptide loops, which bind to different G-proteins, consistent with the distinctive molecular pharmacology of the several EP₃ receptor subtypes/splice variants. Because knowledge of the structural relationships among the different PG receptors has increased rapidly, new avenues of research into the functional roles and relationships among these important proteins are being identified.

The FP receptor, which mediates the biological effects of PGF₂, has been characterized by functional pharmacological methods (Coleman, 1987; Chen et al., 1995), by receptor binding assays (Powell et al., 1976; Woodward et al., 1995) and recently by molecular cloning techniques (Abramovitz et al., 1994; Sugimoto et al., 1994; Sakamoto et al., 1994; Lake et al., 1994). In the absence of selective antagonists, potent and selective agonists, such as fluprostenol and cloprostenol, have been used to classify the FP receptor (Coleman et al., 1994). As with other classes of PGs, there appear to be marked species and tissue differences in the physiological effects of PGF₂. PGF₂ causes luteal regression in many species (Coleman et al., 1990) and has been reported to contract smooth muscle in various tissues, including the gastrointestinal tract, blood vessels and uterus (Coleman et al., 1990, 1994), from which the human FP receptor cDNA was isolated (Abramovitz et al., 1994; Lake et al., 1994). In the eye, PGF₂ and analogs have been shown to lower intraocular pressure in humans and primates, but this effect is not observed in all species (Bito et al., 1983; Wang et al., 1990). The potential of PGF₂ analogs as new therapeutic agents to lower elevated intraocular pressure has led to increased interest in the FP receptor as a target for drug discovery.

Preliminary studies have demonstrated the presence of a PG receptor linked to calcium mobilization in Swiss 3T3 cells (Woodward and Lawrence, 1994; Woodward et al., 1995). However, limited pharmacological studies were performed and no data were presented for coupling of these receptors to the PI turnover signaling mechanism or the pharmacological characteristics of the latter system. The aims of the present study, therefore, were to characterize the pharmacological properties of the PG receptor on the Swiss 3T3 cell line using PI turnover and calcium mobilization bioassays and to correlate these parameters with the pharmacology of [³H]PGF₂ receptor binding to bovine corpus luteum membranes, a tissue highly enriched in FP receptors (Powell et al., 1976; Coleman et al., 1994) and from which the FP receptor was first successfully cloned (Sakamoto et al., 1994). A preliminary account of the present studies has been recently presented (Griffin et al., 1995).

	Materials and Methods

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Cell culture. Swiss albino mouse 3T3 fibroblasts were grown in DMEM containing 4.5 g/liter glucose and 110 mg/liter sodium pyruvate, which was supplemented with 2 mM L-glutamine, 10 µg/ml gentamicin sulfate and 10% fetal bovine serum. Cells were passaged, before reaching confluence, by treatment with 0.05% trpysin/0.53 mM EDTA and were seeded at high dilution for maintenance of the contact-inhibited phenotype (Takuwa et al., 1989). For agonist stimulation experiments, cells were grown to confluence in 24-well uncoated plastic plates.

Receptor binding experiments. The competitive FP receptor binding assay was performed with a bovine corpus luteum membrane preparation (20 mg/ml) incubated with [³H]PGF₂ (0.9-1.5 nM; 150-175 Ci/mmol) and increasing concentrations (in duplicate) of the test compound for 2 hr at 23°C. The nonspecific binding was defined with 1 to 10 µM unlabeled PGF₂. The assays were terminated by rapid vacuum filtration, using Whatman GF/B glass fiber filters that had been previously soaked in 0.3% polyethylenimine, and the receptor-bound radioactivity was determined by liquid scintillation counting at 50% efficiency. The data were analyzed by a nonlinear, iterative, curve-fitting program (Michel and Whiting, 1984; Sharif et al., 1991).

PI turnover experiments. [³H]IPs produced by agonist-mediated activation of PLC were quantified by previously published procedures (Sharif et al., 1994, 1996). Briefly, confluent cells were exposed to 1.0 to 1.5 µCi myo-[³H]inositol (18.3 Ci/mmol) in 0.5 ml of DMEM for 24 to 30 hr at 37°C. Then cells were rinsed once with DMEM/F-12 medium containing 10 mM LiCl, and the agonist stimulation experiment was performed in 0.5 ml of the same medium to facilitate accumulation of [³H]IPs (Berridge et al., 1982; Sharif et al., in press). Cells were exposed to agonist or solvent for 60 min at 37°C (triplicate determinations), followed by aspiration of the medium and immediate addition of 1 ml of 0.1 M formic acid (held at 4°C). The plates were kept cold and then frozen. Samples frozen up to 1 week were thawed before chromatographic separation of radiolabeled components. The cell lysates (0.9 ml) were loaded on columns packed with approximately 1 ml of AG 1-X8 anion-exchange resin. The elution procedure consisted of washes with 10 ml of water, then 8 ml of 50 mM ammonium formate and finally 4 ml of 1.2 M ammonium formate with 0.1 M formic acid, which was collected in a scintillation vial. To this eluate was added 15 ml of scintillation fluid, and the total [³H]IPs were determined by scintillation counting in a beta-counter. Data were analyzed by the sigmoidal fit function of the Origin Scientific Graphics software (Microcal Software, Northampton, MA) to determine agonist potency (EC₅₀ value) and efficacy, relative to the standard cloprostenol.

Studies of intracellular calcium mobilization. The agonist-stimulated mobilization of intracellular Ca⁺⁺ in Swiss mouse 3T3 cells was investigated by the fura-2 fluorescent Ca⁺⁺ chelator method (Grynkiewicz et al., 1985), using stirred suspensions of cells in cuvettes. Some modifications of published procedures (Yamaguchi et al., 1988) were made, as described. To avoid trypsin degradation of the membrane receptors, cells grown in 175-cm² flasks were detached by room temperature incubation with 0.05% EDTA in phosphate-buffered saline without Mg⁺⁺ and Ca⁺⁺, containing 0.1% glucose, for 35 to 45 min. The cell suspension was centrifuged briefly, and the pellet was immediately resuspended in Hanks' BSS containing 1.3 mM Ca⁺⁺. Cells were subsequently washed twice in Hanks' BSS containing 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer, pH 7.4, and 0.1% BSA (designated as BSS-BSA buffer) and were then loaded with 2 µM fura-2/AM in the BSS-BSA buffer at room temperature in the dark for 60 min. Fura-2/AM was removed by washing the cells three times with the BSS-BSA buffer. Cell counts and cell viability (by trypan blue exclusion) were determined, and a concentrated stock suspension of the fura-2-loaded cells in the BSS-BSA buffer was stored on ice in the dark.

Materials. Swiss albino mouse 3T3 fibroblasts (CCL-92, passage 116) were purchased from the American Type Culture Collection (Rockville, MD). Tissue culture reagents and other reagents purchased from Life Technologies (Grand Island, NY) included DMEM, DMEM/F-12 medium, glutamine, gentamicin, trypsin/EDTA, BSS, phosphate-buffered saline without Ca⁺⁺ or Mg⁺⁺, Hanks' BSS and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid. Fetal bovine serum (Hyclone, Logan, UT) was heat-inactivated at 56°C for 30 min and stored at 20°C. EDTA (disodium salt), Tris base, BSA, digitonin, formic acid, ammonium formate, LiCl and polyethylenimine were supplied by Sigma Chemical Co. (St. Louis, MO). Ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N,N-tetraacetic acid was a product of Fluka BioChemika (Buchs, Germany); Molecular Probes, (Eugene, OR) was the source of fura-2, fura-2/AM, the nonfluorescent Ca⁺⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid and Ca⁺⁺/EDTA calibration buffers. Amersham Corp. (Arlington Heights, IL) was the source of myo-[³H]inositol, and [³H]PGF₂ was purchased from DuPont NEN (Boston, MA). 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). U73122 and U73343 were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). All PGs were purchased from Cayman Chemical Co. (Ann Arbor, MI) or synthesized at Alcon by published methods, except as follows: SC-46275 and SC-19220 were kindly provided by G.D. Searle (Skokie, IL), and AH6809 was purchased from Tocris-Cookson (St. Louis, MO).

	Results

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PI turnover and receptor binding experiments. The cloprostenol-stimulated [³H]IPs accumulation in Swiss 3T3 cells at 37°C was linear for at least 1 hr over a range of agonist concentrations (data not shown), and the maximum response did not decrease significantly even at cloprostenol concentrations as high as 100 µM. Therefore, all subsequent studies were conducted at 37°C for 1 hr. The accumulation of [³H]IPs in these cells in response to stimulation by six PGs known to have some degree of selectivity for distinct PG receptors is shown in figure 1. The potencies of these compounds were as follows (for all, n = 3-45): 16-phenoxy-PGF₂ (EC₅₀ = 0.61 ± 0.1 nM), cloprostenol (EC₅₀ = 0.73 ± 0.04 nM), 17-phenyl-PGF₂ (EC₅₀ = 2.71 ± 0.35 nM), fluprostenol (EC₅₀ = 3.67 ± 0.61 nM), PhXA85 (EC₅₀ = 27.3 ± 5.63 nM) and PGF₂ (EC₅₀ = 28.5 ± 5.26 nM) (table 2; fig. 1). In contrast, PGD₂ (EC₅₀ = 155 ± 29.9 nM; E_max = 49% of cloprostenol), PGE₂ (EC₅₀ = 2570 ± 566 nM; E_max = 59% of cloprostenol) and U46619 (EC₅₀ = 1060 ± 310 nM; E_max = 63% of cloprostenol) were less potent and were partial agonists (table 2; fig. 1). Based on these results, cloprostenol was selected as the "standard control agonist" included in all experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent stimulation of [3H]IPs formation in Swiss 3T3 cells by compounds with known selectivity for different PG receptors. Each plot is representative of several such experiments for each agonist. , cloprostenol; , fluprostenol; , PGF2; , PGD2; , PGE2; , U46619. Each point is the average of triplicate determinations in a single experiment, with S.E.M. shown. Data from numerous experiments of this type are shown in table 2.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent displacement of [3H]PGF2 bound to the bovine corpus luteum membrane preparation, by the PG receptor ligands shown in figure 1. Each point represents the mean ± S.E.M. from several experiments (as indicated), with duplicate samples in each experiment. , cloprostenol (n = 4); , fluprostenol (n = 4); , PGF2 (n = 4); , PGD2 (n = 3); , PGE2 (n = 3); , U46619 (n = 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Correlation of ligand binding affinities (log IC50) and functional potencies (log EC50) in the [3H]IPs accumulation assay for a range of PGs. All data for compounds active in both assays (table 2) are included (n = 15). Linear regression analysis yielded a slope of 0.69 and a correlation coefficient of 0.94.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Concentration-dependent effects of U73122 and U73343 on [3H]IPs formation in Swiss 3T3 cells stimulated by cloprostenol. After a 15-min preincubation with varying concentrations of U73122 () or U73343 (), the cells were exposed to cloprostenol (10 nM) for 1 hr. Also shown are the control experiments, with the agonist alone (, U73122; , U73343) or either effector compound alone (, U73122; , U73343). The data shown are from a representative experiment, but the IC50 values obtained from four experiments are presented in "Results."

Intracellular calcium mobilization studies. Studies of agonist-stimulated mobilization of intracellular Ca⁺⁺ in 3T3 cells were undertaken with a limited number of compounds. The intracellular Ca⁺⁺ concentration signal produced by the active agonists was concentration-dependent (fig. 5). The rank order of potency and efficacy of PGs as stimuli of intracellular Ca⁺⁺ mobilization (table 3) was similar to that established in the [³H]IPs production assay (table 2), with FP agonists being more potent and efficacious than agents selective for DP and EP receptors (table 3). However, the most potent agonists that could be discriminated and thus readily rank ordered in the PI turnover assay, i.e., cloprostenol, fluprostenol, 17-phenyl-PGF₂, 16-phenoxy-PGF₂ and PhXA85, had similar potencies (60-70 nM) in the intracellular Ca⁺⁺ mobilization assay.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Concentration dependence of the intracellular Ca++ response of fura-2-loaded Swiss 3T3 cells for several active agonists. The plots shown are representative of several experiments with each compound. , cloprostenol; , fluprostenol; , PhXA85; , PGF2; , PGD2. The mean responses ± S.E.M., where duplicate determinations were made, are shown. Data from several such experiments are shown in table 2.

	Discussion

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We have used the techniques of PI turnover and intracellular Ca⁺⁺ mobilization to pharmacologically define the functionally coupled PG receptors present on Swiss mouse 3T3 fibroblast cells. Because Powell et al. (1976) previously demonstrated the enrichment of FP receptors in the bovine corpus luteum, and because the first FP receptor cloning was successfully accomplished using this tissue (Sakamoto et al., 1994), we compared the pharmacological profile of the 3T3 cell PG receptor with that of the bovine corpus luteum, using receptor binding techniques. The results of all our studies strongly indicate that the sole receptor mediating PLC-induced PI turnover and intracellular Ca⁺⁺ mobilization in the 3T3 cells is the FP receptor. Supportive data for this conclusion include the findings that, whereas prototypic FP agonists (such as cloprostenol, fluprostenol, PHXA85 and 17-phenyl-PGF₂) (table 1) had nanomolar potency and were highly efficacious agonists in both functional assays and receptor binding assays, PGs selective for DP receptors (e.g., BW246C, BWA868C and PGD₂) (table 1), EP receptors (e.g., enprostil, sulprostone, misoprostil, butaprost, 17-phenyl-PGE₂ and 11-deoxy-16,16-dimethyl-PGE₂) (table 1), IP receptors (e.g., iloprost) and TP receptors (e.g., U46619) were of much lower potency and showed significantly lower affinities than the FP ligands. Moreover, the failure of two EP₁ receptor antagonists (SC-19220 and AH6809) (table 1) and a DP antagonist (BWA868C) to inhibit the [³H]IPs response to 17-phenyl-PGE₂ and PGD₂, respectively, indicated that these PGs were also activating the FP receptor in the Swiss 3T3 cells. To further corroborate these conclusions, we recently showed the presence of a strong signal for an FP receptor mRNA transcript in Swiss 3T3 cell lysates, using RT-PCR techniques, and only a very faint transcript band for TP receptor mRNA (N. A. Sharif, M. Senchyna, D. J. Crankshaw and B. W. Griffin, unpublished observations).

Although initial intracellular Ca⁺⁺ mobilization studies in 3T3 cells suggested the potential presence of FP receptors in these cells (Woodward et al., 1990; Woodward and Lawrence, 1994), we have now provided extensive pharmacological evidence, using two different bioassays, to corroborate these earlier findings. Our studies have also indicated that the resolution of agonist potencies and efficacies, and thus the ability to rank order compounds, was much more clearly defined using the [³H]IPs accumulation assay, compared with the intracellular Ca⁺⁺ mobilization assay. Moreover, the role of PLC was conclusively demonstrated by inhibition of the response by U73122, a selective PLC inhibitor (Bleasdale et al., 1990). The inhibition constant of U73122 (1.24 µM) was in the range of published values for various PLC-coupled receptors (Bleasdale et al., 1990). The aforementioned combined information was not previously available in the literature and is an important finding for future use of the Swiss 3T3 cells for pharmacological evaluation of PGs. As mentioned above, although there are limited data demonstrating that PGF₂ produces the expected IP response in both NIH 3T3 and Swiss 3T3 cells (Corps et al., 1989; Nakao et al., 1993), studies of PLC activation in either cell line by other PGs, including known selective FP receptor agonists, have not been reported. Hence, our PI turnover data for the Swiss 3T3 cells represents novel information that is important for the classification of the PG receptor expressed by these cells.

Other studies with "3T3" cells of various types have been reported. Corps et al. (1989) studied the Ca⁺⁺ mobilization responses of single, attached "Swiss 3T3" cells to PGF₂, vasopressin and other peptides. These agonists, at a single high concentration, also stimulated formation of IPs in fura-2-loaded cells. The cellular responses of "NIH 3T3" cells to PGF₂, and binding of [³H]PGF₂ to membranes from these cells, were characterized by Nakao et al. (1993), who attributed some discrepancies between their results and earlier data to clonal variations in the NIH 3T3 cell line. In a study of PGF₂-induced signal transduction in "3T3-L1" fibroblasts, it was reported that vasopressin, bradykinin and bombesin produced no response (Nakada et al., 1990), consistent with our negative results with these agonists in the Swiss 3T3 cells. Although some differences in the particular cells used, as well as the methodology, could account for differences in PGF₂ potency in the different studies, our EC₅₀ values of 28.5 nM and 102 nM for the [³H]IPs accumulation and Ca⁺⁺ responses, respectively, agree more closely with the values reported by Nakao et al. (1993) (EC₅₀ values of 46 nM for [³H]IPs and 75 nM for Ca⁺⁺) than those determined by Nakada et al. (1990) for the 3T3-L1 fibroblasts (250 nM for both responses). Similar potency values (EC₅₀ = 36-45 nM) for PGF₂ were also reported for bovine corpus luteal cell PI turnover and intracellular Ca⁺⁺ mobilization (Davis et al., 1987). Quantitative potency data were not reported in recent publications describing the Ca⁺⁺ mobilization responses of Swiss 3T3 cells in stirred cell suspensions (Woodward et al., 1990; Woodward and Lawrence, 1994), but EC₅₀ values estimated from their graphical data in those publications appear to be generally consistent with our results for PGF₂, fluprostenol, PGD₂ and PGE₂. In the same studies, sulprostone and U-46619 increased intracellular Ca⁺⁺ with potencies estimated to be in the range of 1 to 5 µM and BW245C was inactive, consistent with the quantitative PI turnover data reported in this work.

An excellent correlation between FP receptor functional potency in the Swiss 3T3 cells and binding affinity of several PGs for the bovine corpus luteal FP receptors was observed (fig. 3). The receptor affinity and rank order data for six key PGs, obtained from inhibition of [³H]17-phenyl-PGF₂ binding to Swiss 3T3 cells membranes (Woodward et al., 1995), also correlated well with our functional data in the 3T3 cells and the [³H]PGF₂ binding data from the corpus luteum. Furthermore, our functional and ligand binding data were also well correlated with the rank order of potency of fluprostenol, cloprostenol, PGF₂, PGD₂ and PGE₂, as determined in dog and cat iris contraction assays (Coleman et al., 1990). All of these results are consistent with recent gene sequencing data suggesting that the FP receptor probably exists as a single isoform with a high degree of homology among species (Lake et al., 1994). However, the study of Lake et al. (1994) revealed variability in size of the mouse FP receptor transcripts (2-6 kilobases) and also low levels of a larger transcript (6.5 kilobases) in those human tissues (e.g., ovary) with highest amounts of the 6-kilobase transcript. Based on these results, the possibility of minor subtypes of the FP receptor could not be eliminated. However, the profiles of the concentration-response and concentration-inhibition plots of our functional and receptor binding data indicated the presence of a single FP receptor exhibiting high affinity for PGF₂ analogs in both preparations. Even though there appears to be considerable homology in the FP receptor of various species, there are some notable differences in pharmacological characteristics of this receptor expressed in transfected cells (typically COS cells). As evidence for receptor identity, the ligand binding properties of each of these receptors have been determined with membranes from the transfected cells; these results have demonstrated that PGF₂ binds more strongly than other classes of PGs (Abramovitz et al., 1994; Sugimoto et al., 1994; Sakamoto et al., 1994; Lake et al., 1994). However, the IC₅₀ value of PGF₂ for these various FP receptors ranged from a low of about 2.5 nM (Sugimoto et al., 1994; Abramovitz et al., 1994) to 40 nM (Graves et al., 1995). The COS cell-expressed human FP receptor has comparable affinity for PGF₂ (2.8 nM) and PGD₂ (7.0 nM) and only about 30-fold lower affinity for PGE₂ (85 nM), compared with PGF₂ (Abramovitz et al., 1994). In contrast, the FP receptors of cow, sheep, mouse and rat, expressed and characterized by very similar techniques, have IC₅₀ values of 300 to 500 nM for PGD₂ and 0.4 to 2.0 µM for PGE₂ (Sugimoto et al., 1994; Sakamoto et al., 1994; Lake et al., 1994; Graves et al., 1995). Based on these limited data, the PG binding selectivity of the FP receptor gene product from the several animal sources appears to be similar to that of the bovine corpus luteum FP receptor used in our studies. The significant differences in absolute IC₅₀ values of PGF₂ for the expressed FP receptor genes of different species are unexpected, because the ligand-binding region of this receptor should be among the most highly conserved parts of the protein. Differences in the experimental conditions used to transfect, select and grow the transfected cells could influence the membrane environment of the FP receptor and, consequently, the specific and nonspecific binding characteristics of the FP receptor preparation. Also, the specific activity of the radiolabeled ligand probe determines the minimal detectable signal and the limits of the assay. Although differences in methodology could account for the observed species differences in binding of the FP receptor gene product to PGF₂ and other classes of PGs, other explanations for these results, such as distinct FP receptor subtypes, cannot be excluded at this stage.

In our study, the IC₅₀ value for binding of PGF₂ to the FP receptor of bovine corpus luteum was about 3-fold larger than the largest value reported for the expressed FP receptor gene. As the data in table 1 and figure 3 indicate, the IC₅₀ values determined from the binding assay were about 1 log unit larger than the EC₅₀ values for agonist-dependent PI hydrolysis by Swiss 3T3 cells. Several factors, in addition to those mentioned above, may account for the differences in absolute potency or binding affinity (IC₅₀ value) measured for a given compound in various assays. The sensitivity of each assay is determined by the minimal number of receptors required for an acceptable signal-to-noise ratio (reflecting the inherent "signal" of the radiolabeled probe or fluorescent probe, in the case of the Ca⁺⁺ assay), the degree of amplification of the response by physiological means (activation of an enzyme) or other methods (such as the use of LiCl to inhibit metabolism of IP species) (Berridge et al., 1982), the sources and types of background signals in the different assays and other experimental conditions that may be unique to the particular assay. For example, the PI turnover assay has some unique characteristics, including enzymatic amplification, LiCl-dependent accumulation of the radiolabeled "second messengers" and, as discussed, no apparent contribution from other functional receptors on Swiss 3T3 cells that might bind PGs. Consequently, the signal-to-noise ratio for this assay was excellent, and the responses produced by very low concentrations of very potent agonists could be easily detected with small numbers of cells. It is more difficult to directly compare the potency values for the [³H]IPs accumulation and Ca⁺⁺ responses measured in 3T3 cells, because of the limited number of compounds evaluated in the latter assay. To the best of our knowledge, there are few published articles with extensive comparative potency data generated in the two types of assays. There are multiple physiological controls over the kinetics, magnitude and duration of the intracellular Ca⁺⁺ response that cannot be easily varied by the experimenter. Also, the use of detached cells (in contrast to the attached cell monolayers used in the PI turnover assay), reliance on a chelator-ion association phenomenon and inherent limitations of quantifying fluorescence signals in a biological milieu impose additional constraints on absolute potency values that can be measured by the fura-2 method. Although each assay of receptor "activity" used in this study appears to have somewhat different discriminatory power for the most potent compounds, the rank order of potency of compounds with a wide range of potencies was similar in all assays, again reinforcing the tenets of the pharmacological process to classify the PG receptors.

The quantitative potency and efficacy data obtained in this study confirm that the FP receptor, like other PG receptors, is not absolutely selective for FP agonists and responds to relatively high concentrations of other classes of endogenous PGs and synthetic analogs. PGs are considered to serve mainly as locally produced autocrine and paracrine signals. Consequently, their production by cyclooxgenase 1 and 2 (Laneuville et al., 1994; Mitchell et al., 1994) and their subsequent metabolism are tightly controlled, to ensure that their concentrations are maintained within physiological limits. Also, tissue-specific control over the particular PGs synthesized from arachidonic acid and the identity, density and occupancy of specific PG receptors contribute to the selectivity of prostanoid-mediated physiological effects. Other factors also influence the functional selectivity of the PG receptors, such as the type of G-protein involved in the ensuing signal transduction cascade. The possibility that interaction of the receptor with its G-protein could be a mechanism of control of ligand binding affinity, and thus selectivity, has received experimental support for certain PG receptors (Negishi et al., 1993). As PG receptors and their associated G-proteins are characterized more thoroughly, and as the molecular functions of the cyclooxygenase enzymes are elucidated, our understanding of the mechanisms controlling functional selectivity of different PG classes will greatly increase.

In conclusion, the data in this report have increased our understanding of the in vitro pharmacological properties of the FP receptor and have provided additional evidence that the PG-responsive receptor in Swiss 3T3 cells, which is coupled to PLC activation and intracellular Ca⁺⁺ mobilization, is indeed the FP receptor. Our data demonstrated that an assay for FP receptor function that uses the physiological signal amplification process coupled to receptor activation, e.g., [³H]IPs formation, provided by an intact cellular system is a valuable method to generate quantitative potency and efficacy data for agonists with a broad range of potencies and efficacies. However, as described in this report, ligand binding techniques used in combination with Ca⁺⁺ mobilization and PI turnover studies provided a more thorough characterization of the physiological and pharmacological properties of the FP receptor, and thus such a multidisciplinary approach represents a powerful means to study and classify receptors.