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Am J Physiol Regul Integr Comp Physiol 279: R1495-R1503, 2000;
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Vol. 279, Issue 4, R1495-R1503, October 2000

Expression of members of the multidrug resistance protein family in human term placenta

M. V. St-Pierre1, M. A. Serrano2, R. I. R. Macias2, U. Dubs1, M. Hoechli3, U. Lauper4, P. J. Meier1, and J. J. G. Marin2

Division of Clinical Pharmacology and Toxicology, Departments of 1 Internal Medicine and 4 Obstetrics, University Hospital and 3 Electron Microscopy Laboratory, University of Zürich, CH-8091 Zürich, Switzerland; and 2 Departments of Biochemistry and Physiology, School of Pharmacy, University of Salamanca, 37007 Salamanca, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The placenta serves, in part, as a barrier to exclude noxious substances from the fetus. In humans, a single-layered syncytium of polarized trophoblast cells and the fetal capillary endothelium separate the maternal and fetal circulations. P-glycoprotein is present in the syncytiotrophoblast throughout gestation, consistent with a protective role that limits exposure of the fetus to hydrophobic and cationic xenobiotics. We have examined whether members of the multidrug resistance protein (MRP) family are expressed in term placenta. After screening a placenta cDNA library, partial clones of MRP1, MRP2, and MRP3 were identified. Immunofluorescence and immunoblotting studies demonstrated that MRP2 was localized to the apical syncytiotrophoblast membrane. MRP1 and MRP3 were predominantly expressed in blood vessel endothelia with some evidence for expression in the apical syncytiotrophoblast. ATP-dependent transport of the anionic substrates dinitrophenyl-glutathione and estradiol-17-beta -glucuronide was also demonstrated in apical syncytiotrophoblast membranes. Given the cellular distribution of these transporters, we hypothesize that MRP isoforms serve to protect fetal blood from entry of organic anions and to promote the excretion of glutathione/glucuronide metabolites in the maternal circulation.

placenta transport; human multidrug resistance protein 1; human multidrug resistance protein 2; human multidrug resistance protein 3


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE TERMINAL VILLI of human placenta at term, the barrier between the maternal and fetal blood consists of a single-layered syncytium of polarized epithelial cells, the syncytiotrophoblast, a sparse number of cytotrophoblasts, and the villus stroma, which are separated from the fetal endothelium by a basement membrane (44). The passage of solutes between the maternal and fetal blood in either direction requires translocation across the maternal-facing apical brush-border and fetal-facing basolateral membranes of the syncytiotrophoblast as well as across the endothelium of the fetal capillaries. The removal of end products of metabolism by the placenta is vital when the fetal renal and hepatic systems have insufficient excretory capacities (24). The fetal liver synthesizes and metabolizes organic compounds that undergo minimal biliary excretion, among which are bile salts, anionic amphipathic compounds derived from the catabolism of cholesterol (9). Fetal bile salts are vectorially translocated from the fetal to the maternal circulation for elimination (35). At the apical brush-border membrane of the syncytiotrophoblast, bile salt transport is partially ATP dependent (5, 35), suggesting that a member of the ATP-binding cassette (ABC) superfamily of membrane transporters pumps these compounds in the intervillous maternal blood. At this time, only two ATP-dependent ABC cassette transporters have been localized to the trophoblastic tissue of human placenta: the cystic fibrosis transmembrane conductance regulator (CFTR; see Refs. 14 and 40) and P-glycoprotein multidrug resistance gene product 1 (MDR1; see Refs. 10, 40, 48). CFTR presumably mediates chloride transport at the apical membrane of the syncytiotrophoblast (14). The P-glycoprotein present in apical syncytiotrophoblast may function as an ATP-driven efflux pump to limit exposure of the fetus to toxic cationic xenobiotics. ATP-dependent uptake of vincristine has been demonstrated in membrane vesicles of cytotrophoblasts (42). In P-glycoprotein-deficient mice, the placental MDR1 content correlated with fetal exposure and toxicity to an analog of avermectin, a known substrate of MDR1 (31). The expression of the multidrug resistance protein (MRP) family, which preferentially transports organic anions such as glucuronide and glutathione conjugates (22, 23), has not been studied. Immunohistochemistry studies have reported both the presence (15) and the absence (48) of MRP1 in the syncytiotrophoblast of term placenta. Furthermore, there have been no functional studies associated with their presence in human placenta. Because it is important to predict fetal exposure to a range of xenobiotics present in maternal blood, we investigated the expression of organic anion transporters in the human placenta at term.


    METHODS
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INTRODUCTION
METHODS
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Materials. Radiolabeled 1-chloro-2,4-dinitro-[14C]benzene (Amersham Switzerland) was used to synthesize the radiolabeled glutathione conjugate, dinitrophenyl-glutathione (DNP-SG), according to the method of Ishikawa (21). Glutathione and glutathione S-transferase were from Sigma Chemie. ATP, creatine kinase, and creatine phosphate were from Boehringer. Nitrocellulose filters (0.65 µm) were from Sartorious. [6,7-3H]estradiol-17-beta -D-glucuronide was from New England Nuclear (Boston, MA). Tritiated glycocholate was a gift from Dr. A. Hofmann (La Jolla, CA).

RT-PCR. Total RNA from human placenta was extracted as described by Chomczynski and Sacchi (7). mRNA was isolated with the Poly(A)tract mRNA Isolation System (Promega, Madison, WI). Placental mRNA and human liver mRNA as a positive control were primed with oligo(dT) and reverse transcribed (SuperScript II; Life Technologies). As a negative control, template mRNA was processed without RT. The resulting cDNA was amplified by PCR with several sets of specific primers for the human MRP1, MRP2, and MRP3 nucleotide sequences. For MRP1 (EMBL/GenBank accession number L05628), 25 cycles of PCR with primers 5'-GATCAACGAGTCTGCC-3' (forward) and 5'-CAGCTCTGGGGCTCAC-3' (reverse) followed by nested PCR with primers 5'-GGCATCAACATCGCCAAGA-3' (forward) and 5'-AAGACCTCTCTGCTGCAGG-3' (reverse) yielded a product of 495 bp. For human MRP2 (X96395), the specific primers were 5'-CTGCCTGTTCTTCATCTC-3' (forward) and 5'-TACCCCTGCCTGTTCTTC-3' (reverse), which gave a 1,800-bp PCR product. For human MRP3 (Y17151), the specific primers 5'-CTGCTGATTGAAGACACAC-3' (forward) and 5'-TACTCCTTGACCCTCTCCAC-3' (reverse) gave a PCR product of 1,150 bp. PCR products were ligated (SureClone Ligation Kit; Pharmacia Biotech) into the PUC19 vector and transformed into MaxDH500 competent bacteria (Life Technologies). Sequencing of plasmid inserts was performed on an ALFexpress DNA Sequencer (Pharmacia Biotech). The subcloned RT-PCR products were used as probes to screen a placenta cDNA library (Origene Technologies, Rockville, MD).

Cloning of human placental MRP homologs. The cDNA library panel was screened by PCR with DyNAzyme EXT DNA polymerase (Finnzymes) and the specific primers for MRP1, MRP2, and MRP3. Subplates containing Escherichia coli glycerol stocks were screened with the same primers in a second round. Positive colonies were identified by hybridization with probes radiolabeled with [32P]dCTP by the random-primed method. Inserts were sequenced as described.

Membrane preparations. Membrane vesicles were prepared from healthy placentas before the onset of labor and within 30 min of scheduled caesarian deliveries. Basolateral (fetal-facing) trophoblastic vesicles were purified according to a modified method of Kelley et al. (25, 35). Apical trophoblastic vesicles (maternal-facing) were prepared according to an adaptation (5) of the method of Booth et al. (3). Basolateral vesicles were characterized as described (13), by measuring binding of the beta -adrenergic antagonist dihydroalprenolol to assess enrichment (49) and ouabain binding in the presence and absence of detergent to assess the orientation and integrity of the vesicles (34). The enrichment and orientation of apical vesicles were characterized by alkaline phosphatase activity (3) and the latency of sialic acid to hydrolysis by neuraminidase (2), respectively. Contamination by microsomal membranes was assessed by the activity of NADPH cytochrome c reductase (37). Protein concentrations were determined by the method of Lowry as modified by Markwell et al. (36).

Immunofluorescence and Western blot analysis. The monoclonal antibodies against MRP1 [MRP(m5) and MRP(m6)] and against MRP2 (M2III-6) were from Alexis Biochemicals. The polyclonal antibodies to MRP3, raised in rabbits against a 20-amino acid internal peptide sequence (ALL) and the 24-amino acid carboxyl terminus (FDS), were developed and described by Konig et al. (28). Markers for endothelial cells, rabbit anti-human von Willebrand factor (VWF), and mouse anti-human platelet endothelial cell adhesion molecule (PECAM-1) (clone WM-59) were purchased from Sigma Chemie. C219, a murine monoclonal antibody against MDR1 and MDR3, was from Signet Laboratories (Dedham, MA).

Placentas were dissected free of fetal membranes and frozen in isopentane precooled in liquid nitrogen, and then sections (10 µm) were cut on a cryomicrotome at -20°C. For studies with antibodies MRP1(m6), MRP1(m5), MRP3(ALL), and PECAM-1, sections were fixed in 2% paraformaldehyde in PBS. Acetone fixation (-20°C) was used for studies with MRP3(FDS) and MRP2. Sections were blocked in BSA (Jackson Immunoresearch Laboratories, West Grove, PA) and whole human serum (Dako, Denmark) and then were incubated with antibodies for 2 h at dilutions of 1:10 for MRP1(m5), MRP1(m6), and MRP2, 1:500 for VWF, 1:200 for MRP3(FDS), 1:500 for MRP3(ALL), and 1:100 for PECAM-1. After four washes in PBS, sections were incubated for 45 min with the secondary antibodies, indocarbocyanine (Cy3)-conjugated goat anti-rabbit or anti-mouse IgG (Jackson Immunoresearch), or FITC-labeled sheep anti-mouse or anti-rabbit IgG (ICN Biomedicals). Sections were examined by confocal microscopy (Leica Instruments).

For Western blot analysis, basolateral and apical placental membranes (100 µg) were diluted with sample buffer and heated to 56°C for 10 min before separation on a 7.5% SDS acrylamide gel. After transfer to a nitrocellulose membrane and blocking in 5% milk and 0.4% BSA, immunoblotting was performed with a 1-h incubation with antibodies against MRP1, MRP2, and MRP3(FDS) diluted in Tris-buffered saline containing 0.1% Tween 20. Signals were detected by means of enhanced chemiluminescence (ECL-Plus; Amersham Pharmacia Biotech) using 1:20,000 dilutions of the secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG, or sheep anti-mouse IgG (Bio-Rad, Munich, Germany).

Transport studies in placenta vesicles. The transport studies were performed at 37°C, as described previously (47), in transport medium containing 10 mM HEPES-Tris (pH = 7.4), 250 mM sucrose, 100 mM KNO3, 10 mM MgCl2, 0.2 mM CaCl2, 3 mM ATP, and an ATP-regenerating system (3 mM creatine phosphate and 100 µg/ml creatine phosphokinase). Transport medium (80 µl) was mixed with 20 µl vesicles (100 µg protein), and the reaction mixture was stopped by addition of 4 ml ice-cold stop solution (250 mM KCl, 25 mM MgSO4, and 10 mM HEPES-Tris, pH = 7.4) and then filtered under vacuum through 0.65-µm cellulose-nitrate filters. For the glycocholate studies, 100 µM cholic acid was added to the stop solution. Filters were washed with 2 × 4 ml stop solution, and radioactivity retained on the filter was measured in a liquid scintillation counter.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Amplification of MRP cDNA fragments by PCR. To screen for the presence of MRP-related proteins in term placenta, we performed PCR with specific primers for MRP1, MRP2, and MRP3 and then subcloned and sequenced the resulting PCR fragments. For MRP1, nested PCR was performed to increase the yield of product available for subcloning. All amplified PCR fragments were of the predicted length. The fragments of all three MRPs were 98-99% identical to the known nucleotide sequences.

Characterization of MRP-related cDNA clones in the placenta. To determine whether the three multidrug resistance-associated proteins expressed in the placenta were identical to, or variants of, those identified in other human tissues, a cDNA library panel was screened by PCR. With primers specific for MRP1, the screen gave 14 positive signals of the predicted size (580 bp). The rescreen of one positive subwell resulted in two positive colonies containing inserts of ~4.9 kb. Sequence analysis from the 5'-end showed 100% homology with MRP1 from base number 834 to the poly(A) tail. Similarly, the initial screen of the cDNA panel for MRP2 yielded six signals of ~1,770 bp. The rescreen yielded two colonies with inserts of ~4.9 kb. Sequencing from the 5'-end showed that the partial cDNA was 100% homologous with the published sequence of MRP2 beginning at exon 3 (base number 177). The initial screen of the cDNA panel with primers specific for MRP3 gave 10 positive signals of ~1,160 bp. Rescreening of one of the subplates gave rise to two positive colonies with inserts of ~3.4 kb. The partial cDNA clone was 100% identical to the published MRP3 sequence beginning at base 2000.

Immunolocalization of MRP in the placenta. The MRP1(m6) antibody gave some weak fluorescent staining of the syncytiotrophoblastic layer and the fetal blood vessels (Fig. 1A). The intensity of the fluorescent signal was less in the syncytiotrophoblast and was not observed in all terminal villi examined. An isotype-matched mouse immunoglobulin showed background staining (Fig. 1B). Because others have reported that in a given tissue individual anti-MRP1 antibodies may show disparity in the target and intensity of staining (15), we also examined the fluorescent staining with the MRP1(m5) antibody. Whereas trophoblastic staining was not evident with this antibody, the fetal blood vessels were strongly labeled, as confirmed by double labeling with anti-human VWF, a cytoplasmic marker for endothelial cells (Fig. 1C). The Cy3 [MRP1(m5)] and FITC (VWF) labels were clearly colocalized to the same vessel structure (Fig. 1D). A reconstruction of the serial sections showed, however, that there was no overlap in the signals (Fig. 1E). VWF is located within cytoplasmic vesicles, and this agrees with the punctate cytoplasmic staining observed; the MRP1 staining pattern was continuous and suggested localization at the plasma membrane.


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  		Fig. 1.   Immunofluorescent localization of multidrug resistance protein (MRP) 1 and MRP2 in human placenta. Cryostat sections were incubated with mouse monoclonal anti-MRP1(m6) (A) or a mouse IgG1 control (B) and were visualized with a indocarbocyanine (Cy3)-labeled secondary antibody. The positive signal surrounds the fetal blood vessels and the syncytiotrophoblast layer. C: colocalization of MRP1 using a second anti-MRP1 antibody(m5) (red) and von Willebrand factor (VWF), a marker for the cytoplasm of the endothelial cells (green). Both antibodies labeled the fetal blood vessels. D: higher magnification of a fetal blood vessel from a terminal villus showing continuous membrane staining with anti-MRP1(m5) (red) and discrete punctate cytoplasmic staining with anti-VWF (green). E: extended focus projection of optical sections in the Z plane of the vessel shown in D. There is clear separation of the anti-MRP1 (red) label and the anti-VWF label (green). F: immunofluorescent localization of MRP2 with a mouse monoclonal antibody and a Cy3-labeled secondary antibody. Fluorescence is most prominent in the apical syncytiotrophoblast membrane of terminal villi (white arrow) but rarely visible in the intermediate villi (blue arrow). G: isotype-matched mouse immunoglobulin showed only background staining.

The anti-MRP2 antibody showed clear localization of the staining to the syncytiotrophoblastic layer of the small terminal villi (Fig. 1F). There was only partial or no staining of the larger intermediate villi and no staining of blood vessels. The signal was more intense within the apical membrane, a pattern that was also observed with the anti-MDR1/MDR3 antibody, C219 (data not shown). A control slide incubated with mouse IgG2a showed weak staining (Fig. 1G). The MRP3 (FDS) antibody labeled both the fetal blood vessel structures and the trophoblastic tissue (Fig. 2A). Prior preincubation of the MRP3 antibody with the epitope-specific FDS peptide abolished the staining within the syncytiotrophoblastic layer but not the signals surrounding the fetal capillaries (Fig. 2B). Incubation of sections with preimmune rabbit serum gave no signal (data not shown). To confirm that the MRP3 antibody was indeed recognizing the blood vessel endothelium, sections were fixed in paraformaldehyde and double labeled with the endothelial membrane marker PECAM-1, a cell adhesion molecule whose expression in term placenta has been described by Coukos et al. (11), and the ALL anti-MRP3 antibody (Fig. 2C). Both PECAM-1 and the ALL MRP3 antibody labeled fetal blood vessels, but staining in the syncytiotrophoblast was absent. A control slide incubated with preimmune serum and mouse IgG1 gave a background signal (Fig. 2D). When the immunofluorescence studies with the ALL anti-MRP3 antibody were repeated on sections fixed in acetone, again labeling of fetal vessels was evident (data not shown).


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  		Fig. 2.   Immunofluorescent localization of MRP3 in human term placenta. A: cryostat sections were incubated with a rabbit polyclonal anti-MRP3 antibody and with a Cy3-labeled secondary anti-rabbit antibody. The fluorescent signal surrounded fetal blood vessels and the syncytiotrophoblast layer (arrow). B: preadsorption of the antibody with the antigenic peptide (300 µM) for 1 h displaced the signal in the trophoblast (arrows) but not the blood vessels. C: colocalization of MRP3 (red) and PECAM-1 (CD31; green), a glycoprotein expressed in the endothelial cell membranes. All vessels are labeled with both antibodies. D: control section incubated with rabbit preimmune serum and an isotype-matched mouse immunoglobulin. Only background staining was observed.

Characterization of basolateral and apical vesicle preparations. To confirm the results seen by immunofluorescence and to further localize MRP1, MRP2, and MRP3 to the apical or basolateral membranes of the syncytiotrophoblastic layer, membrane vesicles were prepared for immunoblotting with anti-MRP antibodies. The vesicles were characterized and found to be enriched for conventional markers (Table 1). The apical membranes were enriched 35-fold for alkaline phosphatase, whereas the enrichment of the basolateral membranes was less (13.5-fold), as measured by the binding of dihydroalprenolol (Table 1). The basolateral membranes were slightly more contaminated with the microsomal membrane marker NADPH cytochrome c reductase (6.9- vs. 1.7-fold for basolateral and apical, respectively). The degree of cross contamination between basolateral and apical membranes was similar: 2.9-fold for basolateral in the apical preparation and 2.7-fold for apical in the basolateral preparation. As an additional verification of the membrane preparations, Western blotting was first performed with C219 against P-glycoprotein (Fig. 3A). As expected from previous reports (42), a signal was observed only in the apical membranes. The Western blot of MRP1 demonstrated a signal in the apical membranes with both MRP1(m5) (Fig. 3C) and MRP1(m6) (Fig. 3D). The Western blot of MRP2 showed a distinct signal in the apical membrane preparations (Fig. 3B). The anti-MRP3 FDS antibody was used for immunoblotting of MRP3 (Fig. 4). An immunoreactive band at ~170 kDa was detected in the apical preparation. A second more intense band was prominent at ~60 kDa (Fig. 4A). Both immunoreactive bands were displaced from the apical preparation when the antibody was adsorbed with the antigenic FDS peptide (50 µM) before immunoblotting (Fig. 4B). Interestingly, additional bands appeared in the lane containing the basolateral membranes when the antibody had been preadsorbed.

                              
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  		Table 1.   Characterization of isolated syncytiotrophoblast membranes from human placenta



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  		Fig. 3.   Expression of MRP1 and MRP2 proteins in isolated membranes of human placenta. A: immunoblot of isolated apical (Ap) and basolateral (Bl) syncytiotrophoblast membranes probed with C219 antibody, which detects all MDR P-glycoproteins. Rat liver canalicular membrane was used as positive control. B: immunoblot of apical and basolateral membranes with anti-MRP2 antibody. Positive control was HepG2 cell membranes. A signal at ~180 kDa was observed in lanes containing HepG2 membranes and apical placenta membranes. C: immunoblot of apical and basolateral placenta membranes probed with an anti-MRP1(m5) antibody. Positive control was HepG2 cell membranes. A signal at ~170 kDa was observed in lanes with HepG2 cells and apical membranes.D: immunoblot was performed as described above, except that a second anti-MRP1(m6) monoclonal antibody was used. A positive signal was seen in the apical membrane, and a weaker signal was seen in the basolateral membranes.



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  		Fig. 4.   Immunoblot analysis of MRP3 expression in isolated membranes of human placenta. A: MRP3 expression was detected in isolated apical membranes but not basolateral membrane term placenta using the FDS antibody. B: in the presence of 100 µM FDS peptide, which competed for antibody binding, the signals in the apical membranes at 170 and ~60 kDa were displaced.

Glucuronide and glutathione conjugate transport in the trophoblasts of the human placenta. To correlate the expression of the MRP proteins in the apical membrane of the syncytiotrophoblast layer with function, we tested for the presence of ATP-dependent uptake of an anionic model substrate of MRP1 and MRP2 in apical vesicles. As a positive control for the integrity of the isolated membrane preparation, we first measured the ATP-dependent uptake of the bile salt glycocholate, the transport of which has been well characterized (5, 47; Fig. 5A). Vesicles were also incubated with the [14C]glutathione conjugate DNP-SG in the absence and presence of ATP. As shown in this typical experiment and repeated in three independent preparations, accumulation was clearly ATP dependent (Fig. 5B). Estradiol-17-beta -glucuronide, a natural steroid conjugate whose affinity for MRP isoforms has been well characterized, also exhibited ATP-dependent accumulation in apical syncytiotrophoblast vesicles. When tested at a concentration of 0.5 µM, uptake was 3.1 ± 0.3 pmol · mg-1 · 10 min-1 in the presence of ATP and 2.0 ± 0.1 pmol · mg-1 · 10 min-1 in the absence of ATP.


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  		Fig. 5.   Uptake of [3H]glycocholate (GC; A) and [14C]dinitrophenyl-glutathione (DNP-SG; B) in apical membrane vesicles of human placenta. Vesicles (100 µg) were incubated with substrates at 37°C in the presence or absence of ATP (3 mM) and an ATP-regenerating system. A: 20 µM glycocholate. B: 20 µM DNP-SG. Each point was measured in triplicate ± SD. The experiments were performed in at least 2 separate membrane preparations.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

The MRP family of proteins mediates the ATP-dependent cellular efflux of a wide range of organic anionic compounds. Substrates for MRP1 and MRP2 include bilirubin glucuronosides and other glucuronide, glutathione, and sulfate conjugates (22, 23, 26). The rat Mrp3 has shown preferential ATP-dependent transport of glucuronide rather than glutathione conjugates (17) and of both monoanionic and dianionic bile salts (18). Human MRP3 transports DNP-SG, but its full spectrum of substrates has not been determined (29).

Our findings show that the human placenta at term expresses at least three members of the MRP protein family: MRP1, MRP2, and MRP3. We have isolated partial clones and determined that they are identical to those originally cloned from human lung (8) and liver (28, 43). The main immunoreactivity to MRP1 in term placenta was in the fetal blood vessels of the terminal and intermediate villi. As demonstrated by double labeling with an antibody to VWF, MRP1 was localized to the endothelium of the vessels. The separation of the Cy3 and FITC signals at higher magnification provides evidence that MRP1 is located within endothelial plasma membrane. Other investigators have recently reported the presence of MRP1 in endothelia, including primary cultures of bovine brain microvessels (19), a mouse brain capillary endothelial cell line (30), and in human brain endothelial cells in culture (46). The immunofluorescence study with the MRP(m6) antibody, but not MRP(m5), localized MRP1 to the syncytiotrophoblast, which is in agreement with results of Flens et al. (15) rather than with those of Sugawara et al. (48). Immunoblotting of plasma membrane preparations of the syncytiotrophoblast suggests an apical subcellular localization for this efflux pump.

MRP2 was detected by both immunofluorescence and Western blotting only in the apical membranes of the syncytiotrophoblast and not in fetal blood vessels. Its expression profile therefore differs from that of MRP1 and resembles that of P-glycoprotein, which is also absent from endothelium and present in apical membranes. The MRP3 protein was detected in the syncytiotrophoblast with the FDS antibody. Immunoblotting suggests an apical localization facing the maternal blood. The identity of the second immunoreactive band at ~60 kDa is not known, but its displacement by the immunogenic FDS peptide suggests that a degradative process has occurred during membrane or sample preparation. Many of the small fetal blood vessels were also stained by the FDS antibody, but these were not displaced by the antigenic peptide (Fig. 2). A second anti-MRP3 antibody (ALL) raised against a more proximal region of the protein also stained fetal blood vessel structures. MRP3 colocalized with PECAM-1, an endothelial cell membrane marker whose presence within endothelial cell junctions and at the luminal surface of endothelium in term placenta has been characterized (32). The lack of syncytiotrophoblast staining with the ALL anti-MRP3 antibody on paraformaldehyde-treated sections may reflect loss of antigenicity during tissue fixation.

Because the MRP transporters are important efflux pumps for anionic conjugates in mammalian cells, we hypothesized that apical membrane vesicles from the syncytiotrophoblast would exhibit ATP-dependent transport of model substrates. Our transport studies indeed show that ATP stimulated the uptake of [14C]DNP-SG, a substrate with high affinity for MRP isoforms (26, 27). ATP-dependent uptake of [3H]estradiol-17-beta -glucuronide was also observed. However, this substrate is transported by MRP isoforms with high affinity [Michaelis constant (Km) = 1-7 µM; see Ref. 26] and by MDR1 with much lower affinity (Km = 65 µM; see Ref. 20). Therefore, the rate of transport that we report is a composite of both high- and low-affinity processes and is influenced by relative abundance of the MRP vs. MDR1 proteins.

The physiological function of MRP-related proteins in the syncytiotrophoblast layer remains speculative. It is logical to expect that the apical trophoblastic expression of an ATP-dependent pump capable of transporting organic anions such as sulfate, glucuronide, and glutathione conjugates out of the cell would be well placed to exert a protective role in the human placenta. In particular, MRP2 was shown to enhance resistance of transfected cells to etoposide, cisplatin, doxorubicin, and vincristine (12). In addition, the placenta possesses significant glutathione S-transferase activity, and the pi -isozyme of glutathione S-transferase protein is detectable upon immunoblotting (1, 38). The placenta, therefore, retains the capacity to bioinactivate pharmacologically active molecules and secrete them in the maternal circulation. Likewise, many bile salts produced by the fetal liver are sulfated and amidated (41). Such dianionic sulfated bile salts are substrates for MRP1 and MRP2 (22, 23), and their elimination via maternal blood may be facilitated. In addition to its steroidogenic activity, the placenta also biosynthesizes the polypeptide hormones endothelin-1 and -3, which are presumed to exert both autocrine and paracrine effects in placental tissue (16, 45). Endothelin receptors are present on fetal vessels, on both membranes of the syncytiotrophoblast, and on extravillous cytotrophoblasts (16, 39). Secretion of endothelin-1 from trophoblastic cells in culture has been demonstrated (6). The recent finding that rat Mrp2 is a candidate for the ATP-dependent transport of endothelin-1 (33) raises the possibility of a role for MRP2 in the release and regulation of endothelin-1 levels in placenta.

Perspectives

An exacting study of the presence and function of all MRP-related proteins in human placental throughout gestation is required since at least three additional MRP family transporters have been identified (4, 26). Moreover, a definitive role for MRPs in placental physiology cannot yet be assigned. In particular, the role of MRPs in the fetal capillary placental barrier is not obvious. The endothelial cells of the fetal vessels are continuous, linked by tight and adherent junctions, secrete a basement membrane, and are thought to contribute substantially to total transplacental resistance to solute transfer (32). The paracellular passage of substances such as horseradish peroxidase has been detected, but the accessibility of charged amphiphilic anions across the fetal endothelial cells in either the fetal or maternal directions has not been studied. Although the subcellular location of MRP1 and MRP3 within the endothelial cells could not be ascertained from these studies, it is tempting to speculate that MRPs may exert a protective role by retrieving toxic substances from the fetal blood that have gained access through the paracellular route. Taken together, our findings that MRP isoforms are localized to fetal blood vessels and/or the syncytiotrophoblast of the chorionic villi suggest a role in the vectorial transport capabilities of the placenta. This in turn would serve as a barrier to protect the fetus from noxious substances and would allow the active secretion of endogenous, anionic compounds in the fetal-maternal direction.


    ACKNOWLEDGEMENTS

We thank the Department of Dental Surgery, University of Zürich, for use of their technical facilities and gratefully acknowledge Dr. D. Keppler, Deutsches Krebsforschungszentrum Heidelberg, Germany, for generous gifts of the MRP3 antibodies and for critical review of the manuscript.


    FOOTNOTES

This work was supported by Hartmann Müller-Stiftung für medizinische Forschung Grant no. 726 (to M. V. St-Pierre), by Marie Heim Voegtlin fellowship no. 3234-055037.98 (to M. V. St-Pierre), and by Grants 31-056020.98 (to M. V. St-Pierre) and 31-45536.95 (to P. J. Meier) from the National Science Foundation, Switzerland.

Address for reprint requests and other correspondence: M. V. St. Pierre, Div. of Clinical Pharmacology, Dept. of Internal Medicine, Univ. Hospital, CH-8091 Zürich, Switzerland (E-mail: stpierre{at}kpt.unizh.ch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 January 2000; accepted in final form 21 May 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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