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Expression of the mRNAs and Proteins for the Na⁺/H⁺ Exchangers and Their Regulatory Factors in Baboon and Human Placental Syncytiotrophoblast

Department of Physiological Sciences, Eastern Virginia Medical School (G.J.P., M.G.B., W.A.D.), Norfolk, Virginia 23507; Academic Unit of Child Health, The Medical School and School of Biological Sciences, University of Manchester (C.P.S.), Manchester, United Kingdom; and Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology, Center for Studies in Reproduction, University of Maryland School of Medicine (E.D.A.), Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Gerald J. Pepe, Ph.D., Department of Physiological Sciences, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, Virginia 23501-1980. E-mail: pepegj{at}evms.edu

	Abstract

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In polarized epithelial cells of several organ systems, e.g. the kidney, a family of Na⁺/H⁺ exchangers (e.g. Na⁺/H⁺ exchanger-1 and -3) and their regulatory proteins, Na⁺/H⁺ exchanger regulatory factor and Na⁺/H⁺ exchanger-3 kinase A regulatory protein play a major role in regulating Na⁺/H⁺ exchange integral to cellular homeostasis. Because the primate placenta regulates exchange of Na⁺ and H⁺ between the mother and fetus critical to fetal-placental homeostasis, the current study determined whether Na⁺/H⁺ exchanger-1 and -3 were compartmentalized and associated with expression of Na⁺/H⁺ exchanger regulatory factor and Na⁺/H⁺ exchanger-3 kinase A regulatory protein in baboon and human syncytiotrophoblast. Using RT-PCR, single 413-bp Na⁺/H⁺ exchanger-1 and 190-bp Na⁺/H⁺ exchanger-3 products were expressed by baboon and human syncytiotrophoblasts. The 104-kDa Na⁺/H⁺ exchanger-1 protein was detected by Western blot in microvillus membranes and to a much lesser extent in the basal membranes of the baboon and human syncytiotrophoblasts. In contrast, the 85-kDa Na⁺/H⁺ exchanger-3 protein was detected primarily in membranes contiguous with the basal membranes of the syncytiotrophoblast of both species. Differential localization of Na⁺/H⁺ exchanger-1 and -3 was confirmed by immunocytochemistry. The Na⁺/H⁺ exchanger-3 regulatory protein, Na⁺/H⁺ exchanger-3 kinase A regulatory protein, resided almost exclusively in the basal membranes, whereas Na⁺/H⁺ exchanger regulatory factor was localized primarily to the microvillus membranes in the baboon and human syncytiotrophoblast. Collectively, these results are the first to show that the baboon and human term placental syncytiotrophoblast expressed the mRNAs and proteins for Na⁺/H⁺ exchanger-1 and -3 and their regulatory factors and that Na⁺/H⁺ exchanger-1 and Na⁺/H⁺ exchanger regulatory factor resided primarily in the microvillus membranes, whereas Na⁺/H⁺ exchanger-3 and Na⁺/H⁺ exchanger-3 kinase A regulatory protein were localized to membranes contiguous with the basal membranes and to the basal membranes, respectively. We conclude that a complete Na⁺/H⁺ exchange system is present in the baboon and human term placental syncytiotrophoblast and suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na⁺/H⁺ exchange between the placenta and the maternal and fetal circulations.

	Introduction

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OUR LABORATORIES have shown that there is an estrogen-dependent change in transplacental corticosteroid metabolism that results in maturation of the pituitary-adrenocortical axis critical to primate fetal development (1, 2). In addition, the placenta plays a role in regulating the exchange of Na⁺ and H⁺ between the mother and fetus (3), a process that is also critical for fetal-placental homeostasis and development. Thus, the rate of unidirectional maternofetal Na⁺ transfer across the placenta increases steadily during human pregnancy (4), and the transport of several nutrients (e.g. amino acids) to the fetus is coupled to the transmembrane sodium gradient established across the syncytiotrophoblast (5, 6). Moreover, extrusion of placental H⁺ ion is essential to the maintenance of intracellular homeostasis, and Na⁺/H⁺ exchange is considered the most efficient means of regulating intracellular pH (7), NaCl reabsorption, and cell volume (8).

A family of antiporters known as Na⁺/H⁺ exchangers (NHEs) and their regulatory proteins (7) carries out this array of physiological functions. To date, six NHE isoforms have been identified, and of these, NHE1 and NHE3 have been the most thoroughly studied. The activity of these antiporters is modulated by NHE regulatory factor (NHE-RF) and NHE3 kinase A regulatory protein (E3KARP), both of which are substrates for cAMP-PKA (7, 9). Moreover, to interact with the antiporters, the regulatory factors must first bind to ezrin, a protein of the ezrin-radixin-moesin family, which links the cortical cytoskeleton to the plasma membrane (10). Although antiporter activity has been measured in the microvillus membranes (MVM) and basal membranes (BM) of the human placenta (5, 6, 11, 12), and NHE1 and NHE3 mRNA (13, 14) and proteins (15, 16) have been detected in human term placenta and BeWo cells (17), the localization and expression of these antiporters and their regulatory proteins in primate pregnancy are poorly understood, and experiments to determine regulation in vivo have not been performed. In renal cells, NHE3 gene expression is stimulated by cortisol (18, 19), whereas in MCF-7 cells NHE-RF expression is up-regulated by estrogen (20). We propose, therefore, that a link between estrogen-regulated cortisol metabolism and expression of the Na⁺/H⁺ antiporter system exists within the primate placenta, which plays an important role in Na⁺ and H⁺ exchange between the maternal and fetal compartments to maintain homeostasis. As a prerequisite to testing this hypothesis, in the present study we determined whether NHE1 and NHE3 mRNAs were expressed in baboon syncytiotrophoblast, and whether their proteins were compartmentalized in syncytiotrophoblast membranes and associated with specific membrane expression of the NHE regulatory proteins. Results were compared with findings in the human term placenta to determine the applicability of the baboon for the study of regulation of antiporter expression and impact on fetal development.

	Materials and Methods

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Tissue preparation
Placentas were obtained by cesarean section from baboons on d 165–170 (i.e. late; n = 3) of gestation (term = d 184) and from women (n = 2) with uncomplicated term pregnancies. Protocols were approved by the animal care and use committee and institutional review board of the Eastern Virginia Medical School. After removal of the decidua basalis and chorionic plate, sections of the villous placenta were placed in phosphate-buffered formalin (Fisher Scientific, Fairlawn, NJ) or snap-frozen and stored in liquid nitrogen. Approximately 60–70 g whole villous tissue were then used for preparation of syncytiotrophoblast MVM and BM essentially as described by Kamath and Smith (21) and recently developed for use in baboon placenta (22). Briefly, villous tissue was minced, stirred in PBS (pH 7.4; Amresco, Solon, OH), and filtered through nylon mesh (Sefar America, Inc., Kansas City, MO) to obtain filtrate and particulate fractions. The filtrate was then centrifuged at 800 x g (to pellet debris), 10,000 x g (to pellet intracellular organelles), and then at 150,000 x g, after which the membrane pellet was homogenized in Tris-mannitol-MgCl₂ buffer. The sample was centrifuged at 2,200 x g and then at 150,000 x g to obtain the MVM, which were resuspended in Tris-sucrose buffer. The particulate fraction was washed, sonicated to release membranes contiguous with the basal membrane (BMm), and then sonicated again in EDTA to release the BM, which was obtained by centrifugation. The MVM, 10,000 x g pellet BMm, and BM were aliquoted and stored at -80 C. We previously confirmed that alkaline phosphatase activity was enriched 16-fold in the MVM and was not detectable in the BMm or BM using the procedures outlined above (22).

A section of baboon whole villous placenta was also used to obtain an enriched fraction of syncytiotrophoblast as described previously (23). Briefly, villous placenta was dispersed in HBSS containing collagenase (0.1%), hyaluronidase (0.1%), deoxyribonuclease (0.01%), trypsin inhibitor (0.023%), and FBS (0.1%); strained through Nitex cloth; and centrifuged, and the pellet was resuspended in HBSS and layered over 5–70% Percoll gradients. After centrifugation, the layer containing primarily syncytiotrophoblast (density, 1.014–1.021) was resuspended in HBSS and centrifuged, and total RNA was prepared from the resultant pellets as described below.

Western blot analyses
Western blot analyses of proteins in syncytiotrophoblast membrane fractions were performed essentially as described previously (24, 25). After determination of protein concentration by the bicinchoninic acid procedure (Sigma, St. Louis, MO), Laemmli buffer was added to a final concentration of 1x (26), and all samples were heated at 100 C for 5 min, cooled, and loaded (35 µg protein/lane) onto discontinuous 8% (NHE1, NHE3, and ezrin) or 12% (NHE-RF and E3KARP) SDS-polyacrylamide minigels. Samples were electrophoresed and wet-transferred to Immobilon P (Millipore Corp., Bedford, MA), and the membrane was blocked by incubation with 3% BSA in 50 mM Tris, pH 7.5, containing 150 mM NaCl and 0.05% Tween 20 (Bio-Rad Laboratories, Inc., Richmond, CA). Samples were incubated (1 h) at room temperature with polyclonal antibodies to the human proteins NHE-RF and E3KARP (provided by Dr. Chris Yun, Johns Hopkins University, Baltimore, MD) and to ezrin, NHE1, and NHE3 purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies were diluted in buffer I [50 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20, and 0.05% IGEPAL CA-630] containing 1.5% BSA. Membranes were then incubated with donkey antirabbit IgG horseradish peroxidase-conjugated second antibody (Amersham Pharmacia Biotech, Arlington Heights, IL), and proteins were detected using enhanced chemiluminescent reagent (Amersham Pharmacia Biotech). Preliminary studies confirmed that the second antibody contributed no nonspecific bands at the concentrations employed.

Northern blot and RT-PCR analyses
The expression of the mRNAs for NHE1 and NHE3 in whole villous placenta and syncytiotrophoblast was determined by RT-PCR (27), and expression as well as transcript size in syncytiotrophoblast and fetal and/or adult baboon kidney were verified by Northern blot (28). Briefly, tissues were homogenized in guanidine isothiocyanate-sodium acetate buffer containing 2-mercaptoethanol, extracted with chloroform-isoamyl alcohol (24:1), layered over a 5.7 M cesium chloride gradient, and RNA-pelleted by centrifugation (29). For Northern blot, approximately 3–4 µg polyadenylated [poly(A)⁺] enriched RNA (28) were transferred to a nylon membrane (GeneScreen, NEN Life Science Products, Boston, MA) and prehybridized in buffer containing 50% formamide, 0.1% polyvinylpyrrolidone, 0.1% BSA, 0.1% Ficoll, 2.5 x SSPE (0.375 NaCl, 0.025 M NaH₂PO₄-H₂O, and 2.5 mM EDTA-Na₂, pH 7.4), 1% SDS, 10% dextran sulfate, and denatured salmon sperm DNA (100 µg/ml). Hybridization was performed in fresh buffer for 24 h with 1 x 10⁶ cpm/ml ³²P-labeled probe, prepared (30) by incubation of complementary DNA (cDNA) to rat NHE1 and NHE3 supplied by Dr. Gary Shull (University of Cincinnati Medical Center, Cincinnati, OH) and labeled with 50 µCi [-³²P]deoxy (d)-CTP (3000 mCi/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) and the random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN) to a specific activity of 10⁹ dpm/µg DNA. After hybridization, the membrane was washed under stringent conditions and then exposed to Kodak X-AR film (Eastman Kodak Co., Rochester, NY).

For RT-PCR, oligonucleotide primers synthesized by Life Technologies, Inc. (Gaithersburg, MD), were selected from cDNA sequences specific to human NHE1 (31) and NHE3 (14): NHE1 primer 1: upstream, 5'-CTCCACCGTCTCCATGCAGAACATCC' (position 1803–1828); primer 2: downstream, 5'-CCTTCAGCTCCTCATTCACCAGGTCC' (position 2190–2215); and NHE3 primer 1: upstream, 5'-GGCAGGAGTACAAGCATCTGTACAGC' (position 1883–1908); primer 2: downstream, 5'-TTTCTCCGCTTCTGGGCACGC TCC' (position 2049–2072). Total RNA (2 µg) from baboon and human placental samples and nonpregnant baboon kidney were reversed transcribed at 42 C for 60 min in a reaction mixture (20 µl) containing 1 mM each of dATP, dCTP, dGTP, and dTTP (Promega Corp., Madison, WI); 1 mM dithiothreitol; 200 U Superscript ribonuclease HRT (Life Technologies, Inc.); 40 U RNAguard ribonuclease inhibitor (Pharmacia Biotech, Piscataway, NJ); 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl₂; and 250 ng random primers (Life Technologies, Inc.). Because of the complex secondary structure of baboon and human NHE3 mRNA, the primers were annealed with RNA before RT using avian myeloblastosis virus reverse transcriptase (Promega Corp.) at 58 C. After 60 min, the RT mixture was heated to 70 C for 15 min and then cooled to 4 C. DNA amplification was carried out in a 50-µl reaction volume containing 5 µl RT reaction; 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl₂; 1.25 U cloned Thermus aquaticus DNA polymerase (AmpliTaq, Perkin-Elmer Corp./Cetus, Norwalk, CT); and 20 pmol each of primers 1 and 2. PCR was performed in a programmable thermal cycler (MJ Research, Inc., Cambridge, MA), and the sample was amplified in 30 sequential cycles at 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min. After the last cycle, the sample was incubated for 5 min at 72 C. Two negative controls, in which either RNA or RT was omitted from the reaction, were also performed. An aliquot of the PCR products was fractionated by electrophoresis in a 2% agarose gel and stained in ethidium bromide. The 413- and 190-bp NHE1 and NHE3 PCR target products were visualized with a UV transilluminator and photographed. Both amplified products were purified with QIAGEN (Chatsworth, CA) and sequenced using both upstream and downstream primers by the San Diego State University Microchemical Core Facility (San Diego, CA). The baboon NHE1 amplified product was 99% and 100% homologous to the human NHE1 sequence at the nucleotide and amino acid levels, respectively. The baboon NHE3 amplified product was 98% homologous with the human NHE3 sequence at both the nucleotide and amino acid levels.

Immunocytochemistry
Sections (4 µm) of formalin-fixed paraffin-embedded placentas were preincubated for 15 min in 5% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) and then incubated overnight at 4 C with primary antibodies to NHE1, NHE3, NHE-RF, E3KARP, or ezrin diluted 1:200 to 1:500 in 5% normal goat serum-PBS. Studies were also performed with NHE3 antibody preabsorbed with rat NHE3 protein (Vector Laboratories, Inc.). After washing in PBS, sections were incubated with biotinylated antirabbit IgG (DAKO Corp., Carpinteria, CA), biotin was detected with an avidin-biotin peroxidase kit (Vector Laboratories, Inc.), and sections were lightly counterstained with Gill’s hematoxylin.

	Results

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Amplification of baboon and human syncytiotrophoblast RNA by RT-PCR with NHE1-specific primers generated a single 413-bp product comparable to that amplified using RNA from adult baboon kidney as well as the cDNA to rat NHE1 (Fig. 1A). Northern blot analysis confirmed that the cDNA for NHE1 hybridized to a single mRNA species in baboon fetal and adult kidney and placental syncytiotrophoblast that was slightly greater in size than the single 4.8-kb transcript in adult rat kidney (Fig. 1B). As shown in Fig. 2, the 104-kDa NHE1 protein was detected by Western blot primarily in the MVM of baboon and human term syncytiotrophoblast, where expression greatly exceeded that in the BM and other membrane fractions. A smaller, but less abundant, protein was also detected in the MVM of both human and baboon syncytiotrophoblast. Immunocytochemistry confirmed that the NHE1 protein was expressed in the MVM of the human (Fig. 3A) and baboon (Fig. 3B) term syncytiotrophoblast. Moreover, NHE1 was detected in the BM, as noted by Western blot, and in vascular endothelium of the baboon and human placental inner villous core. Specificity was confirmed by the absence of signal in sections (not shown) incubated without primary antibody.

View larger version (61K): [in this window] [in a new window]   Figure 1. A, Representative RT-PCR of NHE1 in an enriched fraction of syncytiotrophoblast of human and baboon term villous placenta and in the kidney of an adult baboon. Two micrograms of total RNA or 1 ng cDNA to rat NHE1 were reverse transcribed, and 5 µl of the RT samples were amplified for 30 PCR cycles using NHE1 primers. Samples were electrophoresed, and the gels were stained in ethidium bromide and photographed. The NHE1 PCR product size was approximately 413 bp. No product was detected in samples in which RNA or RT was omitted from the reactions (not shown). B, Northern blot of NHE1 mRNA in baboon placental syncytiotrophoblast, fetal baboon kidney, adult baboon kidney, and adult rat kidney.

View larger version (66K): [in this window] [in a new window]   Figure 2. Representative Western immunoblot of the 104-kDa NHE1 protein in fractions of baboon and human term placenta isolated as described in Materials and Methods. Syncytiotrophoblast MVM were isolated by mechanical agitation and differential centrifugation, and the BM were released by sonication and isolated by centrifugation. Proteins were loaded (35 µg/lane) onto discontinuous SDS-polyacrylamide gels, transferred to Immobilon P, and incubated with a polyclonal antibody to human NHE1. Bracketed numbers indicate the locations of 32–73 kDa Mr markers.

View larger version (137K): [in this window] [in a new window]   Figure 3. Immunocytochemical expression of NHE1 (A and B) and NHE3 (C–E) in syncytiotrophoblast of the human (A and C) and baboon (B and D) villous placenta and in the kidney of the adult baboon (E). Sections of whole villous tissue or kidney were incubated with polyclonal antibodies to NHE1 (A and B) or NHE3 (C–E). F, Section of baboon placenta incubated with NHE3 antibody preabsorbed with excess immunizing peptide. Original magnification, x1200 (A–F). IVC, Inner villous core.

View larger version (52K): [in this window] [in a new window]   Figure 4. A, Representative RT-PCR of NHE3 in whole villous tissue and syncytiotrophoblast of human and baboon placenta as well as in adult baboon kidney. Two micrograms of total RNA or 1 ng cDNA to rat NHE3 were reverse transcribed, and 5 µl of the RT samples were amplified for 30 PCR cycles using NHE3 primers. Samples were electrophoresed, and the gels were stained in ethidium bromide and photographed. The NHE3 PCR product size was approximately 190 bp. No product was detected in samples in which RNA or RT was omitted from the reactions (not shown). B, Northern blot of NHE1 mRNA in baboon fetal kidney and adult rat kidney.

View larger version (56K): [in this window] [in a new window]   Figure 5. Representative Western immunoblot of the 85-kDa NHE3 protein in various fractions of baboon and human term placenta. Syncytiotrophoblast MVM and intracellular organelles (10,000 x g; 10K) were isolated by mechanical agitation and differential centrifugation, and BMm were released by sonication. Proteins were loaded (30 µg/lane) onto discontinuous SDS-polyacrylamide gels, transferred to Immobilon P, and incubated with polyclonal antibody to NHE3. Peptide block represents protein in baboon and human syncytiotrophoblast (35 µg/lane) probed with NHE3 antibody preabsorbed with excess immunizing peptide. Bracketed numbers indicate the locations of 32–73 kDa Mr markers.

View larger version (36K): [in this window] [in a new window]   Figure 6. Representative Western immunoblot of E3KARP (46 kDa), NHE-RF (50 kDa), and ezrin (80 kDa) in fractions of baboon and human term villous placenta. Syncytiotrophoblast MVM and BM proteins were loaded (35 µg/lane) onto discontinuous SDS-polyacrylamide gels, transferred to Immobilon P, and incubated with polyclonal antibodies to human E3KARP, NHE-RF, or ezrin. Bracketed numbers indicate the locations of 32–123 kDa Mr markers.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It is well known that the activities of NHE1 and NHE3 are acutely inhibited by cAMP, an effect elicited via the regulatory proteins NHE-RF and/or E3KARP. Using in vitro transfection and a yeast two-hybrid system, Yun et al. (9) showed that E3KARP binds NHE3, whereas Reczek et al. (34) demonstrated that ezrin binds PKA type II. Moreover, Lamprecht et al. (35) showed that E3KARP is a protein adapter between ezrin and NHE3, whereas NHE-RF links ezrin and NHE1. Based on this collective information, it has been proposed (33) that the regulatory proteins E3KARP and NHE-RF indirectly localize PKA type II near NHE3 and NHE1, respectively, thereby providing specificity in the protein kinase signaling pathway by colocalizing protein kinase and its substrates, NHE3 and NHE1. Thus, the results of the present study not only show that a complete Na⁺/H⁺ exchange system is present in the syncytiotrophoblast of the baboon and human term placenta, but also suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na⁺/H⁺ exchange between the placenta and the maternal and fetal compartments.

In most polarized epithelial cells, including those of kidney and intestine, NHE1 is localized primarily to the basolateral membranes, and NHE3 is localized to the brush border (i.e. microvillus) membranes (36, 37, 38). Thus, the distribution of these antiporters in human and baboon placenta differs from that in most other polarized epithelial cells. Moreover, in the single study performed to date comparing Na⁺/H⁺ exchanger activity between MVM and BM (12), it appears that amiloride-sensitive (i.e. NHE1) antiporter activity in the MVM exceeds amiloride-resistant (i.e. NHE3) antiporter activity in the BM of human term placenta. We propose therefore, as illustrated in Fig. 7, that these qualitative and quantitative differential patterns of antiporter localization in the primate syncytiotrophoblast may be physiologically significant, because H⁺ ions, the levels of which increase in association with the increase in metabolic activity of the placenta with advancing gestation, would be secreted primarily into the maternal compartment, which, unlike the fetus, has the compensatory mechanisms (e.g. lung and kidney) capable of preventing acidosis. Moreover, as a result of the exchange of Na⁺ and H⁺ at the MVM, maternal Na⁺, the unidirectional maternofetal flux of which increases with advancing gestation (4), would increase in the placenta and be secreted into the fetal compartment. It is possible, therefore, that alterations in expression of NHE1 and/or NHE3 and/or their regulatory proteins in the syncytiotrophoblast could have profound effects on fetal-placental homeostasis. Because estrogen regulates NHE-RF expression in MCF-7 cells (20), whereas cortisol regulates renal NHE3 levels (18), it is possible that estrogen directly and/or via control of placental cortisol metabolism (2) is linked to and thus impacts on expression of the antiporter system in the primate placenta essential to fetal-placental homeostasis. Considering the similar distribution of the NHE system across the syncytiotrophoblast in the baboon and human, as demonstrated in the current study, the baboon appears to be a particularly useful nonhuman primate model to conduct such clinically relevant regulatory studies.

View larger version (15K): [in this window] [in a new window]   Figure 7. Differential expression of NHE1 and NHE3 and their regulatory factors, NHE-RF and E3KARP, in the MVM and BM of the syncytiotrophoblast of the primate placenta and proposed role of the antiporter system in placental-fetal Na+-H+ homeostasis.

The results of the current study further showed that NHE3 protein was localized in intracellular organelles of the human and baboon syncytiotrophoblast. Consistent with these observations, NHE3, which is typically expressed in abundance in the brush border of renal epithelial cells, was also detected in intracellular organelles of the rabbit kidney (7). Thus, it has been proposed (7) that intracellular stores may provide a functional reservoir of spare transporter that is transferred to the apical membranes in the kidney. Subcellular redistribution has also been proposed to be involved in the acute regulation of NHE3 in the brush border of human colon (39). Whether the latter mechanism(s) is also operative in the primate placenta remains to be determined.

In summary, we show in the present study that baboon and human term placenta expressed mRNAs and proteins for NHE1 and NHE3 and their regulatory proteins, and that NHE1 and NHE-RF were localized primarily to the MVM, whereas NHE3 and E3KARP were localized primarily to membranes contiguous with the BM and the BM fraction, respectively, of the syncytiotrophoblast. Thus, a complete Na⁺/H⁺ exchange system is present in the placental syncytiotrophoblast, which exhibits polarity with respect to the capacity for regulation of Na⁺/H⁺ exchange between the placenta and maternal and fetal circulation. Furthermore, we propose that the baboon provides an excellent model to study the factors regulating antiporter expression during human pregnancy and the potential link of the estrogen-dependent change in placental corticosteroid metabolism to the expression of the antiporter system.

	Acknowledgments

 
The authors sincerely appreciate the secretarial assistance of Ms. Sandra Huband with the manuscript, Mr. Nicholas Zachos with the preparation of the photomicrographs, Ms. Pam Brien with the baboon husbandry, and R. B. Billiar, Ph.D., with advice and assistance with the Western blot analyses. The generous supply of antibodies to NHE-RF and E3KARP provided by Dr. Chris Yun (John Hopkins University, Baltimore, MD) and of cDNAs to rat NHE1 and NHE3 provided by Dr. Gary E. Shull (University of Cincinnati Medical Center, Cincinnati, OH) is greatly appreciated.

	Footnotes

 
This work was supported by NIH Research Grant R01 HD-13294 (to E.D.A. and G.J.P.) and The Wellcome Trust (to C.P.S.).

Abbreviations: BM, Basal membranes; BMm, membranes contiguous with the basal membrane; E3KARP, NHE-3 kinase A regulatory protein; MVM, microvillus membranes; NHE, Na⁺/H⁺ exchanger; NHE-RF, NHE regulatory factor.

Received November 22, 2000.

Accepted for publication April 23, 2001.

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