INTRODUCTION

Prostaglandins are arachidonic acid metabolites that may play a major role as mediators of cellular function. PGF₂ ¹ has diverse physiological actions ranging from being a potent luteolytic agent (1, 2) to causing smooth muscle contraction in the uterus (3, 4), vasculature (5), and gastrointestinal (6) and respiratory tracts (7, 8). PGF₂ induces DNA synthesis and cell proliferation in 3T3 fibroblasts (9, 10). Neuronal astrocytes respond to PGF₂, which may mediate pain transmission (11). Recently, PGF₂ has also been shown to cause hypertrophy of cardiac myocytes and induction of myofibrillar genes, independent of muscle contraction. These observations suggest a role for the eicosanoid during development, in compensatory hypertrophy and/or in recovery of the heart from injury (12).

Recently, PGF₂ analogs have been shown to reduce intraocular pressure (IOP), in patients with glaucoma (13, 14). Although the precise mechanisms involved remain unclear, the effects of PGF₂ analogs on IOP may be attributed, at least in part, to their actions on the ciliary muscle. PGF₂ reduces IOP by increasing the uveoscleral outflow of aqueous humor (15, 16), possibly by reducing the resistance between the ciliary muscle bundles, via an effect on the extracellular matrix (17).

A single PGF₂ receptor (FP) has been cloned from myometrial tissue (18-22). Given that there is evidence consistent with splice variation of the FP (23), as has been described for other prostanoid receptors (24, 25), we wished to address the possibility that a distinct isoform might mediate the actions of PGF₂ in the ciliary muscle. Clarification of the nature of the human ciliary FP and development of an antibody that specifically recognized the receptor protein would facilitate investigation of the effects of PGF₂ and its analogs on IOP.

PGF₂ is formed from arachidonic acid via metabolic transformation sequentially catalyzed by phospholipases, cyclooxygenases, and a specific PGF synthase (26). However, it is now appreciated that a series of PGF₂ isomers, the F₂ isoprostanes, may also be formed in vivo via a free radical-dependent pathway (27-29). It has been speculated that these F₂ isoprostanes may function as incidental ligands at eicosanoid receptors, and, possibly, activate related receptors of their own (30). To date, attention has focused particularly on 8-iso-PGF₂. This compound is a potent vasoconstrictor. It is also a mitogen and may activate human platelets (31-33). Curiously, despite its F prostaglandin configuration, 8-iso-PGF₂ has been shown to activate thromboxane receptors (TPs), and its biological effects are blocked by TP antagonists (31-33).

We now report the cloning of an FP receptor from the human ciliary body (hcb) cDNA library and its localization on the cell membrane. The gene product is identical to that cloned from human uterus (18). Additional to activation by PGF₂, F₂ isoprostanes may ligate the FP. Consistent with the observation that 8-iso-PGF₂ is virtually ineffective in competing for binding of a PGF₂ analog to the ovine FP (22), the hcb-FP is minimally activated by this isoprostane. However, a structurally related F₂ isoprostane, 12-iso-PGF₂, results in a significant, dose-dependent activation of both recombinant and native FPs. Moreover, 12-iso-PGF₂ exhibits receptor specificity as a ligand; it fails to activate the prostacyclin receptor (IP) and only minimally activates TP isoforms. Furthermore, consistent with these observations, 12-iso-PGF_2a also desensitizes FP-mediated responses. Oxidant stress is thought to be a feature of heart failure and ocular diseases (34-36). In such clinical conditions, incidental activation of distinct eicosanoid receptors by isoprostanes may modulate the course of disease or the response to therapy.

EXPERIMENTAL PROCEDURES

Wild type human embryonic kidney (HEK 293) cells and NIH 3T3 cells were from the American Type Culture Collection (Rockville, MD). [-³²P]dCTP, [-³²S]dATP, [-³²P]ATP, myo-[2-³H]inositol, [methyl-³H]thymidine, [³H]PGF₂, Rapid-hyb buffer, Redi-prime random primer labeling kit and iloprost were purchased from Amersham Life Sciences. Human multiple tissue Northern blots were purchased from CLONTECH (Palo Alto, CA). Tissue culture reagents were purchased from Life Technologies, Inc. Dotap, restriction enzymes, and other molecular biology reagents was purchased from Boehringer Mannheim. Ampli-Taq DNA polymerase and dNTPs were purchased from Perkin-Elmer. The anion exchange resin AG 1-X8 (formate form, 200-400-mesh) was purchased from Bio-Rad. U46619, SQ29548, and PGF₂ were purchased from Cayman Chemicals (Ann Arbor, MI). Horseradish peroxidase-conjugated anti-rabbit IgG and fluorescein isothiocyanate-labeled anti-rabbit IgG were purchased from Jackson Immunologicals (West Grove, PA). 9,11-PGF₂ was kindly provided by Dr. Robert Zipkin, Biomol Research Laboratories, Inc. (Plymouth Meeting, PA).

HEK 293 cells were routinely maintained in DMEM with 10% fetal bovine serum, 1% glutamine, and 0.5% penicillin/streptomycin. Stable transformants were maintained in HEK medium with 1.3 mg/ml G418. HEK 293 cells were kept in humidified 5% C0₂, 95% air at 37 °C. NIH 3T3 cells were maintained in DMEM with 10% fetal bovine serum, 1% glutamine, and 0.5% penicillin/streptomycin in humidified 10% CO₂, 90% air at 37 °C.

Phage DNA was prepared from the hcb-cDNA library (kindly donated by Dr. Miguel CocaPrados, Yale University, New Haven, CT) and subjected to PCR. PCR was performed on 200 ng of human ciliary body cDNA with 100 pmol of the FP-specific sense primer 5-TCGAGGACCTGGTGTTTCTAC-3 (18) and a degenerate antisense primer 5-CCAIGGRTCIARDATYTGRTT-3 (I = inosine, R = G/A, D = G/A/T, Y = T/C), 1 × Ampli-Taq buffer, 3 mM MgCl₂, and 0.5 mM dNTPs in a total reaction volume of 100 µl. The samples were subjected to a "Hot Start" as described previously (37), followed by the addition of 0.5 units of Ampli-Taq DNA polymerase. The reaction was then subjected to denaturation at 99 °C for 1 min, annealing at 50 °C for 2 min, and extension at 72 °C for 3 min for 5 cycles, followed by denaturation at 99 °C for 1 mine, annealing at 55 °C for 2 min, and extension at 72 °C for 3 min for 25 cycles. PCR products electrophoresed on a 1% agarose gel revealed the presence of a ~369-bp band, which generated a positive signal when subjected to Southern blot hybridization with a ³²P-labeled FP-specific oligonucleotide, 5-GACTGGGAAGATAGATTTTAT-3 (18). The ~360-bp PCR product was subcloned into pBluescript to isolate the full-length hcb-FP cDNA and was then used as a probe to screen the hcb-cDNA library in -Uni Zap-XR, as described earlier (37). Hybridization was performed in Rapid-hyb buffer at 65 °C for 3 h. Two positive clones were identified, isolated, rescued, and sequenced. The full-length hcb-FP cDNA isolated was ~3.1 kilobase pairs in size, consisting of 151 bp of 5-untranslated region, 1077 bp of open reading frame, and 1867 bp of 3-untranslated region, ending in a poly(A) tail. The open reading frame of the hcb-FP encodes a 359-amino acid protein with seven putative membrane-spanning domains, belonging to the superfamily of G protein-coupled receptors.

Tissue distribution of the human FP mRNA was analyzed on human multiple tissue Northern blots from CLONTECH (Palo Alto, CA) using a ~650-bp BamHI/HindIII fragment of the full-length FP clone random primed with [³²P]dCTP to a specific activity of 3.1 × 10⁹ cpm/µg. The blots were hybridized in Rapid-hyb buffer at 65 °C for 3 h and washed initially with 50 ml of 5 × SSC, 0.1% SDS at room temperature for 30 min and then with four washes of 50 ml of 0.2 × SSC, 0.1% SDS at 60 °C for 30 min. The blots were then autoradiographed overnight at 80 °C.

A ~ 1.8-kilobase pair EcoRI fragment from the full-length hcb-FP cDNA was subcloned into pcDNA3 (Invitrogen, San Diego, CA). The orientation of the insert was verified by restriction digestions. This expression construct (pcDNA3-FP) was then used to transfect HEK 293 cells using Dotap under standard conditions. HEK 293 cells were also transfected with pcDNA3 to serve as a control. The medium was replaced after 6 h with fresh medium containing 1.5 mg/ml G418. Stable transfectants were selected on medium containing 1.5 mg/ml G418 and screened for the expression of the FP by binding to [³H]PGF₂ and second messenger (inositol phosphate; InsP) generation. One clone (HEK-FP), out of 19 clones selected, was chosen for further characterization.

To study the signal transduction properties of the hcb-FP, confluent cultures of HEK-FP cells in 12-well plates were labeled to equilibrium with myo-[2-³H]inositol (2 µCi/ml) for 16-24 h in serum-free DMEM containing 20 mM HEPES, pH 7.5, and 0.5% Albumax. Cells were preincubated in this medium with 20 mM LiCl for 15 min at 37 °C and then stimulated directly by the addition of agonist for 5-10 min at 37 °C. Total InsP formation was measured as described previously (38). Briefly, InsP formation was stopped by aspiration of the medium, addition of 0.75 ml of 10 mM formic acid and incubation at room temperature for 30 min. The solution containing the extracted InsP was neutralized and diluted with 3 ml of 10 mM NH₄OH (yielding a final pH of 8-9) and then applied directly to a column containing 0.7 ml of the anion exchange resin, AG 1-X8. The column was washed with 4 ml of 40 mM ammonium formate, pH 5.0, to remove the free inositol and the glyceroinositol. Total InsPs were eluted with 4 ml of 2 M ammonium formate, pH 5.0. One ml of the eluate was counted with 9 ml of scintillation fluid. Results presented are an average of three to five independent experiments.

Desensitization experiments were performed essentially as described by Opperman et al. (39). Briefly, after incubation with 20 mM LiCl, cells were pretreated with PGF₂, 12-iso-PGF₂, or PBS (control) for 5 min at 37 °C, followed by immediate aspiration of the medium. The cells were then washed twice with 1 ml of 50 mM glycine, 150 mM NaCl, pH 3.0. The cells were then restimulated with PGF₂ or 12-iso-PGF₂ in medium containing 20 mM LiCl for 10 min at 37 °C. The reactions were terminated and InsPs were extracted as described above. Results presented are an average of three independent experiments.

Polyclonal peptide antibodies were raised in rabbits to the sequence GINGNHSLETCET corresponding to the third extracellular loop of the human FP receptor by Research Genetics Inc (Huntsville, AL). The antisera were tested by immunoblotting, using membranes from HEK-FP cells. HEK-FP membranes were prepared from confluent 100-mm dishes as follows. Briefly, cells were washed once with PBS and scrapped into 20 mM Tris, pH 7.4, containing 4 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride. Cells were lysed by sonication on ice, and membrane fractions were collected by centrifugation at 115,000 × g for 1 h at 4 °C. The resulting pellet was resuspended in the same buffer. Membrane proteins (100 µg/lane) were resolved on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The immunoblot was first blocked with 5% milk in TBS-T. FPs were visualized by treating the immunoblots with 1:500 dilution of the crude peptide antisera (in 5% milk/TBS-T) for 1 h at room temperature, followed by horseradish peroxidase-conjugated anti-rabbit IgG (1:5000 dilution). Antigen-antibody complexes were visualized by chemiluminescence.

For immunocytochemistry, cells were grown on chamber slides (Nunc, Napierville, IL) and fixed with 70% methanol, 30% acetone at 20 °C for 10 min, followed by incubation at room temperature for 5 min. The cells were blocked with 2% BSA/PBS and then treated with 1:200 dilution of anti-FP antisera in 0.5% BSA/PBS for 1 h at room temperature, followed by a 1-h incubation with fluorescein isothiocyanate-labeled anti-rabbit IgG (1:500) in 0.5% BSA/PBS. Between each step, slides were washed three times for 10 min each with PBS. Slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined by fluorescence microscopy with a Nikon Microphot FXA microscope.

We measured [methyl-³H]thymidine incorporation into DNA by the method of Nakamura et al. (40) with slight modifications. NIH 3T3 cells were subcultured into 12-well plates. Confluent cultures were washed three times with PBS and then incubated in serum-free DMEM for 10 h. The quiescent cultures were then washed twice with serum free DMEM and stimulated with agonist in the serum-free DMEM for 24 h at 37 °C. [³H]Thymidine (0.5 µCi/ml) was added to the medium in the last 2 h of incubation. The cells were washed twice with ice-cold PBS at the end of the 24-h period and incubated with 1 ml of ice-cold 10% trichloroacetic acid for 10 min on ice to remove the intracellular pool of unincorporated [³H]thymidine. After the removal of the trichloroacetic acid solution, the cells were incubated with 1 ml of 0.5 N sodium hydroxide for 10 min at room temperature. The sodium hydroxide-soluble sample was counted with 9 ml of scintillation fluid. Results presented are an average of three independent experiments.

RESULTS

The hcb-FP encodes an open reading frame of 359 amino acids that is identical to the uterine FP. Northern blot analysis reveals the FP mRNA to be ~5 kilobase pairs in size and highly expressed in the human heart > pancreas > liver, placenta > skeletal muscle > uterus > kidney > small intestine (Fig. 1).

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We generated a mammalian expression construct of the hcb-FP cDNA in pcDNA3 and used this to transfect HEK 293 cells. One of the stable transfectants (HEK-FP), was chosen for detailed characterization.

Activation of the FP by PGF₂ leads to an increase in InsP formation in HEK 293 cells in a dose-dependent manner, reaching a plateau around 1 µM PGF₂. The EC₅₀ for PGF₂-induced InsP formation is 10 ± 1.5 nM (Fig. 2).

Dose response of PGF₂-induced second messenger generation in HEK-FP cells.

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F₂-isoprostanes are isomers of PGF₂ and have been divided into four structural classes (41-43). To test their possible affinity for the FP, we selected members of the first (IPF₂-I), third (IPF₂-III), and fourth classes (8-iso-PGF₂, 12-iso-PGF₂, and 9, 11-PGF₂) (Fig. 3). As seen in Fig. 4, only 12-iso-PGF₂, among these isoprostanes, caused significant activation of the hcb-FP, as observed by InsP formation in HEK-FP cells. To explore the specificity of 12-iso-PGF₂ for the FP, these compounds were also tested for their ability to activate other prostanoid receptors, namely, the IP and the two cloned isoforms of the thromboxane receptor (TP and TP). Iloprost (a prostacyclin analog) induced a ~2.6 ± 0.4-fold increase in InsP formation in HEK 293 cells stably expressing the human IP receptor (44). However, none of the isoprostanes mimicked this response. On the other hand, when tested on HEK 293 cells stably expressing the TP receptor isoforms (45), 12-iso-PGF₂ resulted in 1.6-1.8-fold increase in InsP formation (Fig. 4). In comparison, equimolar concentrations of U46619, a thromboxane agonist, caused 9-10-fold stimulation of InsP formation in these cells. The 12-iso-PGF₂-induced InsP formation via the TP was abolished by the TP antagonist, SQ29548. As demonstrated previously (33), 8-iso-PGF₂ also activates TPs, and this response was also abolished by SQ29548.

Chemical structures of PGF₂ and selected F₂ isoprostanes.

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Isoprostane-induced InsP formation via the prostanoid receptors is dose-dependent. The EC₅₀ for InsP formation by 12-iso-PGF₂ via the FP is 5 ± 0.7 µM, whereas that for 8-iso-PGF₂ is 20 ± 3.4 µM (Fig. 5, Table I). However, the maximal response to 12-epi-PGF₂ is greater than 500% over control, similar to the maximal response to PGF₂, whereas that for 8-iso-PGF₂ is less than 200% of control. By contrast, TPs appear to favor 8-iso-PGF₂ over 12-iso-PGF₂ as a ligand. The EC₅₀ of 8-iso-PGF₂ on the TP receptors is ~ 2.5-5 µM, whereas that of 12-iso-PGF₂ is greater than 50 µM. Furthermore, the maximal response of the TP to 8-iso-PGF₂ is ~700% over control, similar to their response to the thromboxane analog, U46619 (700-900%), whereas their maximal response to 12-iso-PGF₂ is ~400% over control.

Dose response of 8-iso-PGF₂- and 12-iso-PGF₂-induced InsP formation in HEK-FP and HEK-TP cells.

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Table I. InsP formation Receptor EC50 for Inositol Phosphate Formation PGF2 12-iso-PGF2 8-iso-PGF2 U46619 FP 10  ± 1.7 nM 5  ± 0.8 µM 20  ± 3.4 µM ND TP NDa 50  ± 8 µM 2.5  ± 0.8 µM 10  ± 1.3 nM TP ND 70  ± 14 µM 5  ± 1.2 µM 51  ± 8.6 nM Receptor Maximum responses (% control) PGF2 12-iso-PGF2 8-iso-PGF2 U46619 FP 550  ± 20 530  ± 30 225  ± 25 ND TP ND 480  ± 40 650  ± 40 700  ± 35 TP ND 400  ± 50 675  ± 35 900  ± 60 a ND, not determined.

We generated polyclonal peptide antibodies to the third extracellular loop of the FP. Immunoblot analysis of HEK-FP membranes with anti-FP antisera revealed FP to be a broad complex with a molecular weight ranging from 42 to 55 kDa (Fig. 6A). This signal appeared to be specific to FP inasmuch as it was not evident in pcDNA3 vector-transfected HEK 293 cells. Furthermore, the FP signal was competed away by preincubating the antisera with 10 µg/ml of the corresponding peptide. The human anti-FP antiserum also recognizes the native FP in mouse NIH 3T3 cells. Immunocytochemistry of HEK-FP cells reveals FP to be expressed predominantly at the cell surface (Fig. 6B).

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We investigated the ability of FP to undergo agonist-induced rapid homologous desensitization. Although receptor desensitization is a common method of regulation among G protein-coupled receptors (46), there is very little information available on the regulation of FP function by desensitization. Pretreatment of HEK-FP cells with 1 µM PGF₂ for 5 min causes a significant dose-dependent reduction in InsP formation as compared with control cells (Fig. 7A), although membrane receptor protein (Fig. 7B) and whole cell binding (408.8 dpm/10⁶ cells in control group and 419.6 dpm/10⁶ cells in the pretreated group) remain essentially unchanged. The dose response for pretreatment revealed that attenuation of InsP formation was maximal when cells were pretreated with ~1 µM PGF₂, resulting in ~60% desensitization (Fig. 7C). The ability of 12-iso-PGF₂ to cause FP desensitization was also tested on HEK-FP cells. When pretreated with PGF₂ or 12-iso-PGF₂, FP undergoes rapid desensitization to both PGF₂ and 12-iso-PGF₂ (Fig. 8).

A, agonist-induced rapid homologous desensitization of FP. Confluent cultures of HEK-FP cells in 12-well plates were labeled to equilibrium with myo-[2-³H]inositol (2 µCi/ml) for 16-24 h in serum-free DMEM. Cells were treated with 20 mM LiCl for 15 min at 37 °C and then pretreated with vehicle or with 1 µM PGF₂ for 5 min at 37 °C. Cells were then washed with 50 mM glycine, 150 mM NaCl, pH 3.0, and restimulated with varying concentrations of PGF₂ for 10 min at 37 °C. Total InsP formation was measured as described under "Experimental Procedures." B, immunocytochemistry of HEK-FP cells pretreated with PGF₂. HEK-FP cells plated in chamber slides were treated with vehicle (top) or 1 µM PGF₂ (bottom) for 5 min at 37 °C. Cells were then washed with PBS and processed for immunostaining as described under "Experimental Procedures." C, dose response for pretreatment. Confluent cultures of HEK-FP cells in 12-well plates were labeled to equilibrium with myo-[2-³H]inositol (2 µCi/ml) for 16-24 h in serum-free DMEM. Cells were treated with 20 mM LiCl for 15 min at 37 °C and then pretreated with various concentrations of PGF₂ for 5 min at 37 °C. Cells were then washed with 50 mM glycine, 150 mM NaCl, pH 3.0, and restimulated with 100 nM PGF₂ for 10 min at 37 °C. Total InsP formation was measured as described under "Experimental Procedures."

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12-iso-PGF₂-induced rapid desensitization of FP.

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Both PGF₂ and 12-iso-PGF₂ induce InsP formation in NIH 3T3 cells in a dose-dependent manner (Fig. 9A). The EC₅₀ for InsP formation by PGF₂ is 50 ± 8.3 nM. This is comparable to the EC₅₀ of PGF₂ for InsP formation in HEK-FP cells (Fig. 2) and also comparable to the EC₅₀ for InsP formation reported by Nakao et al. (47) in NIH 3T3 cells (~46 nM). In NIH 3T3 cells, PGF₂ causes a dose-dependent increase in mitogenesis, with an EC₅₀ of ~25 ± 3.8 nM (Fig. 9B). This response was also mimicked by 12-iso-PGF₂. The mitogenic response parallels InsP formation, resulting in a maximum of ~3.2-fold increase over basal values.

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DISCUSSION

We have cloned the FP from a hcb cDNA library, a likely target tissue for the efficacy of FP agonists in the treatment of glaucoma. Although the hcb-FP is identical to that cloned from the human uterus, the isolation of only two clones from the ocular source suggests that the FP is not expressed abundantly in the ciliary body. However, it does not rule out the existence of other FP isoforms (23) in other parts of the eye. Studies on the distribution of FP over a wide range of human tissues reveal its mRNA to be abundant in the human heart, in addition to reproductive tissues, as reported previously (19-21). This is particularly interesting in light of recent reports on the ability of PGF₂ to cause hypertrophy of cardiac myocytes (12). Generation of HEK cells stably expressing the FP presents a tool for the detailed molecular characterization of FP. This is of importance, because there is a discrepancy between the rank order of potency of PGF₂ analogs in their ability to reduce IOP and their ability to bind FP in various membrane preparations (48). As with other prostanoid G protein-coupled receptors (45), the pattern of fluorescence observed with an FP-specific antibody suggests that FP is localized predominantly at the cell membrane. Availability of a human FP-specific antibody will facilitate determination of the pattern of FP receptor expression in the eye.

The molecular mechanisms of agonist-induced rapid FP receptor desensitization have not been elucidated to date. FPs are down-regulated in astrocytes after prolonged (>4 h) exposure to PGF₂ (49), and constriction of bovine sphincter muscle evoked by PGF₂ is down-regulated upon pretreatment of the preparation with the eicosanoid for 45 min (50). We now demonstrate that stimulation of HEK cells expressing FP, or of NIH 3T3 cells expressing endogenous FP, with PGF₂ results in rapid desensitization, initially without loss of receptor protein from the cell surface. The availability of these reagents is likely to facilitate investigation of the mechanism of action of PGF₂ analogs in ocular disease and of tachyphylaxis to FP agonists in the treatment of glaucoma.

F₂ isoprostanes are free radical-catalyzed products of arachidonic acid (28). Up to 64 different isomers may be formed theoretically, belonging to four structural classes (41-43). Initially, these compounds are formed in situ on the cell membrane, from which they may be cleaved by the action of phospholipases to circulate and, ultimately, be excreted in urine (28). Specific measurement of isoprostanes in affected tissues, circulating lipoproteins, and urine holds promise as an approach to study oxidative stress in vivo. A more controversial issue is whether F₂ isoprostanes, or indeed analogous isomeric forms of other eicosanoids (29), might mediate some of the functional consequences of free radical generation. It has been speculated that in their esterified form, they may contribute to free radical-catalyzed membrane injury (28).

The biological effects of isoprostanes have only recently been investigated. Much attention has been paid to one member of the class IV F₂ isoprostanes, 8-iso-PGF₂. This has been shown to stimulate inositol phosphate formation and DNA synthesis in cultured rat aortic smooth muscle cells (32). It is also a potent vasoconstrictor, at least in the renal and pulmonary circulations (51). It also stimulates mitogenesis and modulates platelet function, facilitating aggregation by subthreshold concentrations of conventional platelet agonists, such as ADP and thrombin (33). These effects of 8-iso-PGF₂ are blocked by pharmacological TP antagonists. However, the concentration of 8-iso-PGF₂ needed to evoke these effects seem much greater than that which circulates in vivo (33). Furthermore, 8-iso-PGF₂, unlike other isoprostanes, may also be formed by a cyclooxygenase-dependent pathway (52).

We have recently synthesized several F₂ isoprostane isomers (43, 53-55). One of these, 12-iso-PGF₂ (43), activates the FP in a specific and saturable manner. It seems likely that 12-iso-PGF₂ may be an abundant member of the F₂ isoprostane family, inasmuch as free radical cyclization rules predict that upon formation of a cyclopentane ring, after oxidative modification of arachidonic acid, the adjacent substituents formed are cis to each other. Thus, cyclization of the hydroperoxy radical derived from 11-hydroperoxyeicosatetraenoic acid would lead predominantly to the formation of cis products such as 8-iso-PGF₂ and 12-iso-PGF₂. Two reports actually predict the formation of 12-iso-PGF₂ type products in larger amounts than 8-iso-PGF₂, as a result of such free radical cyclization (56, 57).

Clearly, discrete isoprostanes might activate their own specific receptors. However, despite much speculation, no such receptors have been cloned to date and, save for the case of 8-iso-PGF₂ (which may also be formed enzymatically), specific receptors for the by-products of lipid peroxidation may seem unlikely. A more plausible concept is that isoprostanes act, in concert, as incidental ligands at prostanoid receptors. However, the comparative dose-response relationships for individual isoprostanes versus the natural prostanoid ligand, as exemplified in this report, reveals that highly concentrated forms of isoprostane delivery would be required for membrane receptor activation. Given the coordinate formation of multiple isomeric species, other considerations may pertain. For example, multiple isoprostanes might activate distinct eicosanoid receptors, which culminate in a common biological response. To address this possibility, we explored the capability of two structurally distinct members of class IV isoprostanes, 8-iso-PGF₂ and 12-iso-PGF₂, to activate the FP and TP isoforms, distinct receptors which mediate common biological responses, such as vasoconstriction and mitogenesis. 12-iso-PGF₂ and 8-iso-PGF₂ activate the FP and TP isoforms, respectively. However, neither compound activated the IP, which mediates vasodilation. Thus, F₂ isoprostanes may act cooperatively to facilitate a common biological response via distinct eicosanoid receptors. Isoprostanes may also desensitize the response of eicosanoid receptors to their natural ligand. We have shown previously that 8-iso-PGF₂ may cross-desensitize TPs. Similarly, we now demonstrate that 12-iso-PGF₂ may cross-desensitize the human FP.

In summary, we have cloned a human ocular FP, generated HEK 293 cells stably expressing the ocular FP, and demonstrated membrane localization of the FP protein in these cells. The availability of these reagents will facilitate investigations into the molecular basis of action of PGF₂ analogs in reducing IOP. Furthermore, we have demonstrated that the FP may be activated and desensitized, not only by its natural ligand, PGF₂, but also by F₂ isoprostanes like 12-iso-PGF₂. These observations raise the possibility that the therapeutic response to PGF₂ analogs may be modulated by F₂ isoprostanes in syndromes of oxidant stress, such as glaucoma or congestive heart failure.