Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Prostaglandins (PGs) are evanescent biological mediators, which exert their effects by binding to specific receptors on cells in the immediate vicinity of their production. PGF₂ has diverse physiological actions in vitro. For instance, it causes vascular smooth muscle contraction (Csepli and Csapo, 1975), hypertrophy of cardiac myocytes (Mentz et al., 1988; Karmazyn, 1989; Kunapuli et al., 1998), and is critical to the induction of labor and parturition in vivo (Sugimoto et al., 1997). The actions of PGF₂ are mediated via a membrane receptor, the FP, which belongs to the G protein-coupled receptor (GPCR) superfamily. The FP activates phospholipase C in a pertussis toxin-insensitive manner (Gusovsky, 1991; Nakao et al., 1993; Quarles et al., 1993), suggesting interaction with member(s) of the Gq family of GTP-binding proteins (G proteins). Membrane receptors for PGs may also be activated by isoprostanes (Audoly et al., 2000), free radical-catalyzed PG isomers (Lawson et al., 1999). For example, the FP is activated in a specific and saturable manner by the isoprostane 8,12-iso-iPF₂-III (Kunapuli et al., 1998), previously known as 12-iso-PGF₂ (Rokach et al., 1997).

Despite the absence of enzymatic activity in plasma capable of oxidizing PGF₂ to inactive metabolites, PGF₂ does not activate the FP on cells distant from its site of generation. Like PGE₂ and PGD₂, PGF₂ is cleared in a single passage through any of several vascular beds, such as the lung (Schuster, 1998). Although a carrier-mediated prostaglandin transport has long been suggested (Ferreira and Vane, 1967; McGiff et al., 1969; Piper et al., 1970), only one prostaglandin transporter (rat PGT) has been cloned from a rat library (Kanai et al., 1995). Subsequently, the human analog (hPGT) has been cloned from a kidney cDNA library (Lu et al., 1996) and its gene has been characterized (Lu and Schuster, 1998). The hPGT is a 643 amino acid protein, with 12 putative membrane-spanning domains (Lu et al., 1996). It is widely expressed (Lu et al., 1996) and is induced by laminar shear stress in endothelial cells (Topper et al., 1998). Both the hPGT and the rat PGT take up PGF₂, PGE₂, PGE₁, and PGD₂ with high affinity (Itoh et al., 1996; Lu et al., 1996), whereas they do not transport the prostacyclin analog iloprost (Lu et al., 1996). The stable hydrolysis product of thromboxane A₂, thromboxane B₂, is transported with low affinity (Itoh et al., 1996; Lu et al., 1996). Although it has been suggested that the hPGT imports PGs for intracellular termination of their effects, it may also be involved in the export of newly synthesized PGs to act on membrane GPCRs (Chan et al., 1998).

Given that extracellular PGF₂ may ligate the FP or be imported via PGT, we investigated whether FP activation regulates hPGT activity. We report that FP activation by its cognate ligand PGF₂ and, to a lesser extent, by the isoprostane 8,12-iso-iPF₂-III, inhibits intracellular prostanoid import by the hPGT. This regulation seems to be mediated via the heterotrimeric G protein G_s, but independent of an increase in cyclic AMP levels or cyclic AMP-dependent protein kinase activation.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. All the cell culture media and G418 were purchased from Life Technologies Inc. (Gaithersburg, MD). Zeocin was purchased from Invitrogen (Carlsbad, CA). The anion exchange resin AG 1-X8 (formate form, 200-400 mesh) and 30% acrylamide/bisacrylamide solution were purchased from Bio-Rad (Hercules, CA). [³H]PGF₂ (212 Ci/mmol), [³H]PGE₂ (159 Ci/mmol), myo-[2-³H]inositol (18.0 Ci/mmol), cyclic AMP radioimmunoassay, and enhanced chemiluminescence kits were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). PGF₂ was obtained from Cayman Chemicals (Ann Arbor, MI). Cholera toxin and pertussis toxin were obtained from List Biological Laboratories Inc. (Campbell, CA). N-[2-([p-bromocinnamyl]amino)ethyl]-5-isoquinolinesulfonamide (H89 dihydrochloride) and dibutyryl cyclic AMP (dBcAMP) were obtained from Calbiochem (La Jolla, CA). 3-Isobutyl-1-methylxanthine (IBMX) was obtained from Sigma (St. Louis, MO) and 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid, disodium salt hydrate (DIDS) was purchased from Aldrich (Milwaukee, WI). Iloprost was purchased from Schering-Plough (Berlin, Germany). Anti G_s, G_i, G_q/11, G₁₂, and G₁₃ antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture and Transfection. The human FP was cloned in our laboratory (Kunapuli et al., 1997). A nine-amino-acid hemagglutinin epitope (HA) (YPYDVPDYA) was inserted between the N-terminal initiator methionine and the second amino acid, as described previously for the HA-IP (Smyth et al., 1996). The HA-FP cDNA was subcloned into the BamHI/EcoRI sites of the mammalian expression vector pcDNA3.1 (Invitrogen) and used for stable transfection of HEK293 cells (HA-FP cells). The cDNA encoding hPGT, kindly donated by Victor L. Schuster of the Albert Einstein College of Medicine (Bronx, NY), was subcloned into the HindIII-NotI sites of pcDNA3.1 or pcDNA3.1/Zeo (Invitrogen). The hPGT in pcDNA3.1 was used for stable transfection of HEK293 cells (hPGT cells), whereas the hPGT in pcDNA3.1/Zeo was used for transfection of HA-FP cells. Subcloning into pcDNA3.1/Zeo allowed us to use zeocin as a second selection marker. Cells overexpressing both the HA-FP and the hPGT are indicated as HA-FP/hPGT cells. A G_s dominant negative cDNA (A³⁶⁶S/G²²⁶A/E²⁶⁸A) in pcDNAI was obtained from American Type Culture Collection, Manassas, VA). The minigene constructs in pcDNA3.1 were kindly donated by Drs. Annette Gilchrist and Heidi E. Hamm (Northwestern University, Chicago, IL).

2

Inositol Phosphate Formation. Inositol phosphate formation was measured in HA-FP/hPGT, hPGT, and NIH3T3 cells. Cells were plated in six-well plates at 2.5 × 10⁵ cells/well and, the following day, incubated with 2 µCi/ml of myo-[2-³H]inositol for 20 to 24 h in inositol-free DMEM. The day of the experiment, the medium was replaced with the same medium containing 20 mM LiCl, and the cells were stimulated with different concentrations of PGF₂. HA-FP/hPGT cells were stimulated for 10 min, whereas hPGT and NIH3T3 cells were stimulated for 30 min. Reactions were terminated by aspiration of the medium and inositol phosphates were extracted with 750 µl of 10 mM formic acid for 30 min, as described previously (Vezza et al., 1996; Habib et al., 1997). Agonist-stimulated inositol phosphate formation is expressed as a percentage of the vehicle-treated sample.

Uptake of [³H]PGF₂ Uptake of [³H]PGF₂ or [³H]PGE₂ was carried out as described previously (Lu et al., 1996). HA-FP/hPGT and NIH3T3 cells were plated in six-well plates at 2.5 × 10⁵ cells/well 1 day before the experiment. The hPGT cells did not adhere firmly to the wells. Thus, they were plated 3 to 4 days before the experiment, to allow them to adhere and spread.

2

2

2

2

2

2

2

2

2

Cyclic AMP Measurement. HA-FP/hPGT cells were plated in six-well plates at 2.5 × 10⁵ cells/well. Cells were incubated with IBMX (0.5 mM) for 5 min and then stimulated with different concentrations of PGF₂, or its vehicle, for 10 min at 37°C. HA-FP cells were seeded at 1.2 × 10⁵ cells/ml in 12-well plates and incubated, or not, with pertussis toxin 250 ng/ml for 20 to 24 h. Cells were then stimulated with different concentrations of PGF₂, or its vehicle, for 10 min in the presence of IBMX (0.5 mM). HA-IP cells (Smyth et al., 1996) were plated in 12-well plates at 1.2 × 10⁵ cells/ml and incubated with cholera toxin for 20 to 24 h and stimulated with different concentrations of iloprost (10 pM-10 nM) for 10 min at 37°C.

Immunoblotting. A polyclonal peptide antibody was raised in rabbits to a 17 amino acid sequence of the carboxyl-terminal tail of the hPGT (H-RVKKNKEYNVQKAAGLI-OH) (Research Genetics Inc., Huntsville, AL). Immunoblotting was performed as described previously (Vezza et al., 1996). Briefly, blots were incubated for 1 h with the anti-hPGT antibody diluted 1:2000 in Tris-buffered saline (50 mM Tris-HCl, 250 mM NaCl, pH 7.4) containing 0.1% Tween 20 and 5% milk. Anti-G_s, G_i, G_q/11, G₁₂, and G₁₃ antibodies were diluted 1:200 in the same buffer. A peroxidase-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) was used as secondary antibody and was diluted 1:5000 in the same buffer. Positive bands were revealed by enhanced chemiluminescence.

Statistical Analysis. All results are presented as mean ± S.E.M. Statistical analysis was performed by Student's t test. A p value of 0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

We carried out functional experiments in NIH3T3 cells to determine whether the FP and the PGT might be coexpressed in the same cells. PGF₂ (1 nM-3 µM) induced a dose-dependent increase of inositol phosphate with an EC₅₀ value of 52.5 ± 0.15 nM. The maximal response (4.09 ± 2.4-fold above the vehicle-treated sample, n = 6 experiments in duplicate), was attained at 1 µM PGF₂, demonstrating FP expression in NIH3T3 cells, consistent with previous data (Kunapuli et al., 1997). Expression of PGT in NIH3T3 cells was demonstrated measuring uptake of [³H]PGF₂. Uptake was time-dependent and was inhibited by the anion transporter inhibitor DIDS (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   NIH3T3 cells express PGT. Uptake of [3H]PGF2 was measured at different time intervals and was inhibited by preincubation of the cells for 15 min with the anion transporter inhibitor DIDS (1 mM). Results are expressed as fold of increase above basal (time zero) and are the mean ± S.E.M. of seven (in the absence of DIDS; control) and four (in the presence of DIDS) experiments performed in duplicate. *p < 0.05, compared with control.

We developed HEK293 cells stably expressing the hemagglutinin-tagged FP (HA-FP) together with the hPGT (HA-FP/hPGT cells) to study the regulation of hPGT by FP. We also developed cell lines expressing the hPGT alone (hPGT cells) or the HA-FP alone (HA-FP cells). hPGT expression in several clones of HA-FP/hPGT and hPGT cells was verified by immunoblotting. Two clones were selected for further experiments and uptake of [³H]PGF₂ in these clones confirmed hPGT expression. [³H]PGF₂ uptake after 10-min incubation was 159.7 ± 13.0 dpm/µg of protein in HA-FP/hPGT cells, and 27.1 ± 2.4 dpm/µg of protein in hPGT cells; absolute dpm values ranged between 8,000 and 30,000 dpm/ml in HA-FP/hPGT cells, and between 700 and 1700 dpm/ml in hPGT cells.

Untransfected HEK293 and HA-FP cells did not take up PGs. In fact, the low level of radioactivity associated with untransfected cells or with HA-FP cells incubated with [³H]PGE₂ was not diminished by incubation with DIDS. In addition, the radioactivity associated with HA-FP cells was similar to that associated with untransfected HEK293 cells. For example, the radioactivity measured in HEK293 after 20-min incubation with [³H]PGE₂ was 0.57 and 0.45 dpm/µg of protein in the absence and in the presence of DIDS, respectively. In HA-FP cells, we measured 0.66 and 0.71 dpm/µg of protein in the absence and in the presence of DIDS, respectively. In contrast, [³H]PGE₂ uptake at 10 min was inhibited by DIDS 1 mM by 93 to 96% in HA-FP/hPGT cells.

We checked FP expression in HA-FP/hPGT and in hPGT cells. As expected, HA-FP/hPGT cells express the FP, as demonstrated by an increase of inositol phosphate production upon stimulation with PGF₂ for 10 min (Fig. 2). By contrast, this response was absent in hPGT cells stimulated for up to 30 min with PGF₂ (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   HA-FP/hPGT, but not hPGT cells, express the FP. HEK293 cells were stably transfected either with the hPGT alone (hPGT cells) or with the hPGT together with the HA-FP (HA-FP/hPGT cells). Expression of the FP in HA-FP/hPGT cells was verified by measuring inositol phosphate production upon stimulation with different concentrations of PGF2 for 10 min. No increase in inositol phosphate production was detected in hPGT cells incubated for 30 min with the same concentrations of PGF2. Results are expressed as a percentage of the corresponding vehicle-treated sample and are the mean ± SEM of four (for HA-FP/hPGT cells) and five (for hPGT cells) experiments performed in duplicate.

We measured [³H]PGF₂ uptake in HA-FP/hPGT cells to determine whether stimulation of the FP modifies hPGT activity. Cells were stimulated with different concentrations of PGF₂, washed, and incubated with [³H]PGF₂ to measure uptake of this prostaglandin. Preincubation of the cells with PGF₂ caused a dose-dependent inhibition of [³H]PGF₂ uptake (Fig. 3A). Similar results were obtained when using [³H]PGE₂ in place of [³H]PGF₂ to assess transporter activity (data not shown). Because residual nonradioactive PGF₂ could influence the uptake results, we measured the levels of PGF₂ in the washings by mass spectrometry. Surprisingly, we found residual, detectable PGF₂ even after washing the cells 10 times (7 and 3 nM after incubation of the cells with PGF₂ 100 nM and 1 µM, respectively). Although a dilutional effect by cold PGF₂ might have confounded our results and cannot be completely excluded, the high levels of PGF₂ after the first wash (15 and 202 nM after incubation with PGF₂ 100 nM and 1 µM, respectively) do not parallel the relatively small inhibition of uptake. In addition, the inhibitory effects of added PGF₂ on transport were similar after washing the cells once versus 10 times, despite a substantial difference in the residual concentrations of this compound at these time points.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A, stimulation of HA-FP/hPGT cells with PGF2 inhibits [3H]PGF2 uptake. Cholera toxin (CTX), but not pertussis toxin (PTX), blocks the FP-dependent regulation of hPGT function. HA-FP/hPGT cells were incubated for 20 h with cholera or pertussis toxin (250 ng/ml) or left untreated (control). The following day, cells were washed, stimulated with different concentrations of PGF2, or its vehicle, washed again, and incubated with [3H]PGF2 to assess uptake. Results are expressed as a percentage of the corresponding vehicle-treated sample and are the mean ± S.E.M. of five experiments performed in duplicate. B, stimulation of hPGT cells with PGF2 does not inhibit [3H]PGF2 uptake. Cholera toxin (CTX) and pertussis toxin (PTX) do not have any effect. hPGT cells were incubated for 20 h with cholera or pertussis toxin (250 ng/ml) or left untreated (control) and experiments were carried out as described in A. Results are expressed as a percentage of the corresponding vehicle-treated sample and are the mean ± S.E.M. of four experiments performed in duplicate.

We also evaluated uptake of [³H]PGF₂ in HA-FP/hPGT cells pretreated with PGE₂. PGE₂ activates the FP with a potency roughly 50% lower than PGF₂, as demonstrated by measurement of inositol phosphate production in HA-FP/hPGT cells (4.4-fold increase over basal with 1 µM PGF₂ and 2.3-fold increase with 1 µM PGE₂). On the other hand, PGE₂ is imported by the hPGT with an affinity comparable with that of PGF₂ (Lu et al., 1996). Preincubation of HA-FP/hPGT cells with PGE₂ 1 µM inhibited [³H]PGF₂ uptake less efficiently than the same concentration of PGF₂ ([³H]PGF₂ uptake: 75.5 ± 2.3% of vehicle-treated sample with PGE₂ versus 51.4 ± 1.3% with PGF₂, n = 3 experiments performed in duplicate), indicating that the regulation of the hPGT is receptor-mediated.

Inhibition of [³H]PGF₂ uptake after stimulation of the FP with PGF₂ was also evident in NIH3T3 cells. These results were poorly reproducible (data not shown), possibly due to the low PGT expression. Indeed, we were able to detect PGT expression in NIH3T3 cells only by ribonuclease protection assay, but not by less sensitive techniques, such as Northern or Western blotting (data not shown).

Because the FP is a GPCR, we investigated the possibility of G protein-dependent regulation of the hPGT by the FP. Immunoblotting revealed that both HEK293 and NIH3T3 cells express at least G_s, G_i, G_q/11, G₁₂, and G₁₃ (data not shown). To assess whether G_s or G_i might be involved in hPGT regulation, we repeated uptake experiments in HA-FP/hPGT cells preincubated for 20 h with 250 ng/ml cholera or pertussis toxin. Cholera toxin completely abolished the inhibitory effect of PGF₂ whereas pertussis toxin, in parallel experiments, did not have any effect on the inhibition of transporter activity induced by stimulation of the cells with PGF₂ (Fig. 3A). The effect of cholera toxin on regulation of transporter activity was dose-dependent (Table 1) and concentrations higher than 5 ng/ml were necessary to counteract the FP-mediated inhibition of prostaglandin uptake. For example, dose-response curves to PGF₂ in the absence or in the presence of 1 ng/ml cholera toxin were superimposable (data not shown). To test the hypothesis that the effect of cholera toxin under our experimental conditions is due to G_s down-regulation (Mochly-Rosen et al., 1988; Chang and Bourne, 1989; Boehm et al., 1996), we measured cyclic AMP levels in cells stably overexpressing the hemagglutinin-tagged PGI₂ receptor (HA-IP cells), which activates adenylate cyclase through G_s. Cyclic AMP levels increased from 0.01 ± 0.003 pmol/10⁴ cells (n = 4) to 2.26 ± 0.14 pmol/10⁴ cells (n = 4) in cells stimulated with 10 nM iloprost and not treated with cholera toxin. By contrast, cyclic AMP was not increased above the basal levels in cells pretreated with 250 ng/ml cholera toxin (0.01 ± 0.0006 pmol/10⁴ unstimulated cells versus 0.02 ± 0.003 pmol/10⁴ cells when stimulated with 10 nM iloprost, n = 4). We also measured cyclic AMP levels in HA-IP cells treated with different concentrations of cholera toxin (1-100 ng/ml) and stimulated with 0.1 nM iloprost. Even at 1 and 5 ng/ml, cholera toxin markedly inhibited the agonist-stimulated increase in cyclic AMP (from 69.1 ± 5.5-fold over basal in the absence of cholera toxin to 13.0 ± 0.7- and 10.0 ± 0.02-fold over basal after pretreatment with 1 and 5 ng/ml cholera toxin, respectively).

View this table: [in this window] [in a new window]   TABLE 1 Low doses of cholera toxin do not counteract FP-mediated regulation of hPGT HAFP/hPGT cells were incubated for 20 to 24 h with different concentrations of cholera toxin (CTX, 5-50 ng/ml) or left untreated. The following day, cells were washed, stimulated with PGF2 1 µM, or its vehicle, washed again, and incubated with [3H]PGF2 to assess uptake. Results are expressed as a percentage of the corresponding vehicle-treated sample and are the mean ± S.E.M. of three to five experiments performed in duplicate.

To determine whether ligation of the FP activates G_s, we measured cyclic AMP formation in HA-FP or HA-FP/hPGT cells stimulated for 10 min with different concentrations of PGF₂. Pretreatment of the cells for 5 min with 0.5 mM IBMX was necessary to detect a cyclic AMP increase under these experimental conditions. PGF₂, at concentrations that inhibit hPGT-mediated [³H]PGF₂ uptake, increased cyclic AMP levels, irrespective of whether the cells had been pretreated with pertussis toxin. PGF₂ increased cyclic AMP levels 2.94 ± 0.43- and 3.21 ± 0.72-fold over basal (n = 7) at 100 nM and 1 µM, respectively, in HA-FP cells not pretreated with pertussis toxin (basal = 0.07 ± 0.006 pmol of cyclic AMP/10⁴ cells). The increase in cyclic AMP levels was 3.31 ± 0.28 and 5.45 ± 0.62 fold over basal (n = 9) with PGF₂ 100 nM and 1 µM, respectively, in cells preincubated with 250 ng/ml pertussis toxin (basal = 0.06 ± 0.006 pmol of cyclic AMP/10⁴ cells). An increase in cyclic AMP, even in the presence of pertussis toxin, which inhibits G_i, implies that PGF₂ activates G_s to stimulate adenylate cyclase. Enhancement of receptor-mediated stimulation of cyclic AMP in pertussis toxin-treated cells or membrane preparations has already been reported (Hazeki and Ui, 1981; Katada et al., 1982).

We then investigated whether stimulation of the FP with its cognate ligand regulates hPGT through cyclic AMP-dependent protein kinase activation. HA-FP/hPGT cells were preincubated for 30 min with the stable cyclic AMP analog, dBcAMP, or with the cyclic AMP-dependent protein kinase inhibitor, H89. Neither stimulation nor kinase inhibition had any major effect on PGF₂-mediated inhibition of hPGT activity (Table 2). We verified the effectiveness of H89 as a cyclic AMP-dependent protein kinase inhibitor in in vitro phosphorylation experiments. At 10 µM, H89 completely suppressed histone H1 phosphorylation by cyclic AMP-dependent protein kinase (data not shown). In addition, we did not observe phosphorylation of hPGT in HA-FP/hPGT cells treated with dBcAMP or stimulated with PGF₂ (data not shown) indicating that hPGT regulation is not accompanied by cyclic AMP-dependent protein kinase-mediated phosphorylation and that this kinase does not play an important role in hPGT regulation.

View this table: [in this window] [in a new window]   TABLE 2 Cyclic AMP-dependent protein kinase does not play a major role in hPGT regulation HA-FP/hPGT cells were incubated for 30 min with the kinase inhibitor H89 (10 µM) or with the cyclic AMP analog dBcAMP (10 µM). Control samples were treated with the vehicle. After 30 min, different concentrations of PGF2 or its vehicle, were added and incubated for 10 min. Cells were then washed and incubated with [3H]PGF2 to assess uptake. Results are expressed as a percentage of the corresponding vehicle-treated sample and are the mean ± S.E.M. of five (for control and H89-treated cells) or four (for dBcAMP-treated cells) experiments performed in duplicate.

In addition to cells expressing both HA-FP and hPGT, we developed a cell line expressing only the hPGT (hPGT cells) that was used as a negative control. Indeed, hPGT cells do not express the FP to a detectable extent, as reflected by a lack of stimulation of inositol phosphate synthesis by PGF₂ (Fig. 2). Thus, as expected, stimulation of hPGT cells with PGF₂ did not have any effect on hPGT activity, irrespective of pretreatment with cholera or pertussis toxin (Fig. 3B). In addition, neither H89 nor dBcAMP had any effect on [³H]PGF₂ uptake in hPGT cells (data not shown).

We investigated whether FP stimulation with the isoprostane 8,12-iso-iPF₂-III would have effects similar to those of PGF₂ on hPGT regulation. This compound, previously known as 12-iso-PGF₂ (Rokach et al., 1997), also activates the FP (Kunapuli et al., 1997). 8,12-iso-iPF₂-III influenced [³H]PGF₂ uptake only at 100 µM or above (Fig. 4), consistent with its lower potency as an FP ligand than PGF₂ (Kunapuli et al., 1997). 8,12-iso-iPF₂-III caused a modest increase in cyclic AMP production in HA-FP/hPGT cells at 1 and 10 µM (up to 2.35 ± 0.18 and 1.23 ± 0.17 pmol/well with 10 and 1 µM 8,12-iso-iPF₂-III, respectively, versus a basal level of 0.55 ± 0.17 pmol/well, n = 3) after 30-min incubation in the presence of IBMX. In parallel experiments, 1 µM PGF₂ increased cyclic AMP up to 3.03 ± 0.4 pmol/well (n = 3), again consistent with the isoprostane acting as a weak FP agonist. Measurement of inositol phosphate production in HA-FP/hPGT cells stimulated for 10 min with 8,12-iso-iPF₂-III confirmed that this isoprostane is roughly 100 times less potent than PGF₂ in activating the FP. Indeed, inositol phosphate production increased 2.7-fold over basal in cells stimulated with 1 µM PGF₂, and 2.1- and 2.6-fold over basal in cells stimulated with 10 and 100 µM 8,12-iso-iPF₂-III, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Stimulation of HA-FP/hPGT cells with 8,12-iso-iPF2-III slightly inhibits [3H]PGF2 or [3H]PGE2 uptake. HA-FP/hPGT cells were stimulated with 8,12-iso-iPF2-III, or its vehicle, for 10 (closed symbols) or 30 min (open symbols). Cells were washed and incubated with [3H]PGF2 or [3H]PGE2 for 10 min to assess uptake. Similar results were obtained when using [3H]PGF2 or [3H]PGE2. Results are expressed as a percentage of the corresponding vehicle-treated sample and are from one experiment performed in duplicate, representative of two to seven experiments. PGF2 (1 µM) was tested in parallel experiments as a positive control.

We studied HEK293 cells transiently transfected with hPGT and HA-FP to demonstrate that FP-mediated inhibition of hPGT function occurs in cells other than the particular HA-FP/hPGT clone that we selected for the majority of the experiments. Cells were stimulated with PGF₂ for 30 min before measurement of [³H]PGF₂ uptake and PGF₂-dependent inhibition of the hPGT was again demonstrable (Fig. 5). In parallel experiments, we transiently transfected HEK293 cells with hPGT and HA-IP (Smyth et al., 1996). PGI₂, unlike PGF₂, is not transported via the PGT, although its receptor also couples to Gs activation (Coleman et al., 1994). HEK293 cells transfected with hPGT and HA-IP were stimulated with the PGI₂ analog iloprost, incubated for 30 min before measurement of [³H]PGF₂ uptake. Iloprost did not have any effect on hPGT activity under these conditions (Fig. 5), despite causing a marked increase in cyclic AMP levels (up to 354.2 ± 47.4 and 235.0 ± 12.5 pmol/well with 100 and 1 nM iloprost, respectively, versus a basal level of 3.77 ± 0.29 pmol/well, n = 3).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Prostacyclin receptor (HA-IP) does not regulate [3H]PGF2 uptake. HEK293 cells were transiently transfected with hPGT together with HA-IP (HA-IP + hPGT) or with HA-FP (HA-FP + hPGT). Two days after transfection, cells were stimulated with different concentrations of iloprost or PGF2, respectively, or their vehicle. Results are expressed as a percentage of the corresponding vehicle-treated samples and are the mean ± S.E.M. of five experiments performed in duplicate.

We used a G_s dominant negative construct (Iiri et al., 1999) and a G_s minigene construct (Rasenick et al., 1994; Gilchrist et al., 1999) to implicate further G_s in FP-mediated hPGT regulation. The former has been shown to inhibit human chorionic gonadotropin-stimulated cyclic AMP accumulation in transfected COS7 cells (Iiri et al., 1999), whereas the latter inhibits -adrenergic activation of G_s (Rasenick et al., 1994). HEK293 cells were transiently transfected with HA-FP, hPGT, and the construct of interest. Cells were stimulated 2 days later with PGF₂ and incubated with [³H]PGF₂ to evaluate transporter activity.

Although the Gs dominant negative was not expressed at high levels, as determined by immunoblotting, transfection of cells with this construct (Fig. 6) or the G_s minigene (Fig. 7) blunted the inhibition of transporter activity after FP stimulation. On the other hand, expression of G_i or G_i random order minigene constructs failed to influence FP-mediated inhibition of transporter activity (Fig. 7).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   FP-mediated inhibition of [3H]PGF2 uptake is blunted by expression of a Gs dominant negative construct. HEK293 cells were transiently transfected with hPGT, HA-FP, and either the empty vector or a Gs dominant negative construct, as described under Experimental Procedures. Two days after transfection, cells were stimulated with different concentrations of PGF2. Results are expressed as a percentage of the corresponding vehicle-treated samples and are the mean ± S.E.M. of five (when using 1 and 10 nM PGF2) or nine (when using 100 nM or 1 µM PGF2) experiments performed in duplicate. *p = 0.05, compared with vector-transfected cells.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   FP-mediated inhibition of [3H]PGF2 uptake is blunted by expression of Gs, but not of Gi or Gi in random order (Gi R) minigene constructs. HEK293 cells were transiently transfected with hPGT, HA-FP, and either the empty vector or minigene constructs, as described under Experimental Procedures. Two days after transfection, cells were stimulated with 100 nM and 1 µM PGF2. Results are expressed as a percentage of the corresponding vehicle-treated samples and are the mean ± S.E.M. of six to eight experiments performed in duplicate. *p = 0.011, compared with vector-transfected cells.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Prostaglandins are charged organic anions at physiological pH, thus they may transverse biological membranes inefficiently (Bito and Baroody, 1975; Kanai et al., 1995; Lu et al., 1996). Although initial hydrolysis and subsequent oxidation rapidly inactivate both PGI2 and thromboxane A₂, PGE₂, PGD₂, and PGF₂ are metabolized intracellularly. Thus, it is assumed that termination of the activity of these PGs in vivo is due to uptake followed by intracellular oxidation (Schuster, 1998). Although a carrier-mediated transport for PGs has been hypothesized since the 1960s and multidrug resistance proteins that are transporters for leukotrienes have been characterized (Cole and Deeley, 1998), a PGT has been cloned only recently (Kanai et al., 1995; Lu et al., 1996). The PGT mediates both the uptake (Lu et al., 1996) and the efflux (Chan et al., 1998) of PGs, thus it may be important both in termination of PG effects, and in extracellular release of newly synthesized PGs for ligation of membrane GPCRs.

Untransfected HEK293 cells and cells transfected only with the HA-FP failed to take up [³H]PGF₂. This indicates that expression of the FP per se does not influence [³H]PGF₂ uptake, and that possible internalization of the ligand-bound receptor does not contribute significantly to the intracellular levels of [³H]PGF₂. On the other hand, engagement of the receptor with its cognate ligand dose-dependently regulated import of both PGF₂ and PGE₂ when the hPGT was present.

Given that cold PGF₂ is not completely removed by washing the cells before the incubation with [³H]PGF₂, we cannot completely exclude a dilutional effect of the residual cold ligand. On the other hand, the amount of residual PGF₂ does not correlate with inhibition of uptake, suggesting that a receptor-mediated effect on transporter activity does indeed exist. This hypothesis is strengthened by the following observations: PGF₂ did not influence transporter activity in hPGT cells, which do not express the HA-FP; and PGE₂ regulates the hPGT in HA-FP/hPGT cells with a potency lower than PGF₂, consistent with the lower potency of PGE₂ as an FP agonist.

Activation of the FP by a structurally distinct ligand, the isoprostane 8,12-iso-iPF₂-III, also regulated transporter function. Isoprostanes are free radical-catalyzed products of arachidonic acid and may act as incidental ligands at membrane receptors for prostanoids in vivo (Audoly et al., 2000). Although the isoprostane is a less potent FP agonist, both PGF₂ and 8,12-iso-iPF₂-III activate phospholipase C and adenylate cyclase, presumably via Gq and Gs, respectively. The two ligands can also activate divergent signaling pathways, at least in cardiac myocytes (Kunapuli et al., 1998).

To address the role of G proteins in receptor-transporter interactions, we first examined the effects of pertussis and cholera toxin, probes for Gi and Gs, respectively. Pertussis toxin ADP-ribosylates the  subunit of G proteins of the Gi family when the  subunit is bound to GDP. Thus, pertussis toxin stabilizes G_i in the inactive conformation. For this reason, pertussis toxin is widely used as an inhibitor of G_i (Simon et al., 1991). Regulation of G_s by cholera toxin is complex. ADP ribosylation of G_s by cholera toxin stabilizes the GTP-bound conformation of G_s and decreases its intrinsic GTPase activity (Casey and Gilman, 1988; Freissmuth et al., 1989). This leads to a persistent activation of this G protein, thereby enhancing adenylate cyclase stimulation and production of intracellular cyclic AMP. Prolonged incubation of the cells with cholera toxin, on the other hand, results in down-regulation of G_s with loss of this G protein from the plasma membrane (Mochly-Rosen et al., 1988; Chang and Bourne, 1989; Boehm et al., 1996). When used to probe FP-dependent pathways, cholera toxin, but not pertussis toxin, blocked the effect of FP activation by PGF₂ on transporter function in a dose-dependent manner. These results suggest involvement of Gs in the interaction of the FP with the transporter. Prolonged incubation of cells with cholera toxin has been described to cause G_s down-regulation (Mochly-Rosen et al., 1988; Chang and Bourne, 1989; Boehm et al., 1996) and, as a consequence, reduced hormone-stimulated cyclic AMP production (Mochly-Rosen et al., 1988). Consistent with this, we observed that the PGI₂ analog iloprost is less efficacious in stimulating an increase in cyclic AMP in HA-IP cells treated for 20 to 24 h with cholera toxin. Although cholera toxin, at concentrations as low as 1 ng/ml, markedly decreased iloprost-stimulated cyclic AMP, doses of the toxin higher than 5 ng/ml were necessary to counteract the effect of PGF₂ on [³H]PGF₂ uptake in HA-FP/hPGT cells. Although cholera toxin may have effects unrelated to G_s, these results distinguish dose-dependent regulation of adenylate cyclase from that of the hPGT. Indeed, interaction of the FP with the transporter seems to be largely independent of its ability to catalyze adenylate cyclase activation. Neither pharmacological activation nor inhibition of cyclic AMP-dependent protein kinase modified FP-mediated regulation of transporter function. In addition, marked activation of the cyclase via Gs coupled to the PGI₂ receptor also failed to regulate transporter function. Finally, ligation of the FP with PGF₂ fails to result in phosphorylation of the transporter, despite regulation of its function.

Although these observations do not support a role for Gs-mediated cyclase activation in the regulation of FP-dependent inhibition of transporter function, additional experiments do support the importance of Gs in this phenomenon. Others have shown that cholera toxin causes persistent activation of G_s by inhibiting its intrinsic GTPase activity (Casey and Gilman, 1988; Freissmuth et al., 1989); thereafter, it causes a loss of G_s from the plasma membrane (Mochly-Rosen et al., 1988; Chang and Bourne, 1989; Boehm et al., 1996). Thus, the effects of the toxin might be attributable to adenylate cyclase activation, excluded for reasons mentioned above, or be due to G_s down-regulation. Results obtained by specific inhibition of G_s with dominant negative constructs support the latter contention. By contrast, a minigene construct directed against G_i failed to modify FP-regulated hPGT function, as did pertussis toxin. Although the mechanism through which FP-mediated Gs activation regulates hPGT functions is presently unknown, the kinetics of the reaction suggest that a protein-protein interaction might be responsible for hPGT regulation. Additional experiments would be required to determine whether Gs interacts directly with hPGT, or whether other proteins mediate this interaction.

In conclusion, engagement of the FP by its cognate ligand PGF₂ and, to a much lesser extent, by the isoprostane 8,12-iso-iPF₂-III dose-dependently inhibits import of PGF₂, potentially for metabolic inactivation. Should this system operate in vivo, it might serve initially to amplify rapidly the effects of high local concentrations of PGF₂, which would subsequently result in receptor desensitization. Prostanoids have been shown to augment more gradually their formation by induction of cyclooxygenase-2 (Barry et al., 1997). Although the details by which receptor activation regulates transporter function remain to be elucidated, our data suggest that interaction of the receptor with the transporter is dependent on G_s, apparently functioning in a role independent of its capacity to catalyze activation of adenylate cyclase.