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Endocrinology Vol. 147, No. 6 2986-2996
Copyright © 2006 by The Endocrine Society

Developmental Regulation of the Sodium/Hydrogen Ion Exchangers and Their Regulatory Factors in Baboon Placental Syncytiotrophoblast

Gerald J. Pepe, Marcia G. Burch and Eugene D. Albrecht

Department of Physiological Sciences (G.J.P., M.G.B.), Eastern Virginia Medical School, Norfolk, Virginia 23501-1980; and Departments of Obstetrics/Gynecology/Reproductive Sciences and Physiology (E.D.A.), The Center for Studies in Reproduction, University of Maryland School of Medicine, 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
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although Na+/H+ exchange is important to maintenance of pH and volume and thus placental-fetal homeostasis, regulation of the Na+/H+ exchange system is incompletely understood. We previously showed that Na+/H+ exchanger (NHE)1 and -3 and their regulatory factors NHERF1 and -2 were expressed in human and nonhuman primate placenta. Our laboratories have also shown that estrogen regulates key aspects of primate placental function and development including the 11ß-hydroxysteroid dehydrogenase enzymes controlling cortisol metabolism. Therefore, it is possible that localization and/or expression of components of the syncytiotrophoblast NHE system are also estrogen dependent. As a first step in testing this possibility, the current study compared the immunocytochemical localization and level of NHE1, NHE3, NHERF1, and NHERF2 in baboon placentas obtained at mid- (d 100) and late gestation (d 165; term = d 184). NHE3 and NHERF2 were abundantly expressed at midgestation and localized to the cytoplasm and juxtanuclear compartment but were not detected in the microvillus membrane. By late gestation, NHE3 and NHERF2 expression were markedly reduced in the juxtanuclear compartment but not the cytoplasm. NHERF2 was also abundantly expressed in fetal vascular endothelium in which levels, as assessed by immunoblot exhibited a 3-fold developmental increase. In contrast, levels of NHE1 and NHERF1, which were abundantly expressed in and localized almost exclusively to syncytiotrophoblast microvillus membrane, were similar at mid- and late gestation. We conclude that the subcellular distribution and levels of key components of the Na+/H+ system in the baboon syncytiotrophoblast are developmentally regulated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL established that the primate placenta plays a significant role in regulating the exchange of Na+ and H+ between mother and fetus, a process that is critical to fetal-placental homeostasis and development (1, 2). For example, the transport of several nutrients to the fetus is coupled to the transmembrane sodium gradient established across the syncytiotrophoblast (3, 4) and Na+/H+ exchange is considered a most efficient means of regulating intracellular pH (5) as well as cell volume (6). A family of antiporters known as Na+/H+ exchangers (NHEs) and their regulatory factors (NHERFs) carries out this array of physiologic functions (7). We previously demonstrated that the mRNAs and proteins for NHE1 and NHE3 and their regulatory proteins NHERF1 and NHERF2 are expressed in baboon and human term placenta (8). Moreover, we showed that near term, NHE1 and NHERF1 protein resided primarily in the syncytiotrophoblast microvillus membranes (MVM), whereas NHERF2 was localized to the basal membrane (BM)/fetal vascular endothelium (FVE). NHE3 expression was most abundant in the cytoplasm and presumably associated with intracellular organelles, which in kidney cells have been identified as recycling endosomes that reside in the cytoplasm and juxtanuclear compartment and serve as reservoirs for and from which NHE3 moves to the cell membrane (9, 10). We proposed, therefore, that a complete Na+/H+ exchange system is present in the primate placenta and that the syncytiotrophoblast near term exhibits polarity with regard to the capacity for regulation of Na+/H+ exchange between the placenta and the maternal and fetal compartments (8).

Fetal growth rate and requirements for particular solutes (e.g. calcium) change during the course of gestation (2, 11). Moreover, to enable the acceleration in fetal growth characteristic of the second half of gestation, placental transfer changes with advancing pregnancy. For example, cationic amino acid transport is greater in human placental MVM vesicles isolated at late than midgestation (12, 13). Our laboratories have shown that estrogen regulates key aspects of placental development and function (14) including the switch between mid- and late gestation in localization and expression of the 11ß-hydroxysteroid dehydrogenase (11ß-HSD) enzymes (15, 16) that controls increased metabolism of cortisol (17, 18), a steroid hormone shown to regulate NHE3 expression (19, 20). Therefore, it is possible that the localization and/or level of expression of key components of the placental Na+/H+ exchange system are regulated directly and/or indirectly by estrogen. As a first step to examine this possibility, we used our baboon model to determine whether levels and/or sites of expression in the syncytiotrophoblast of components of the NHE system were developmentally regulated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female baboons (Papio anubis) weighing 12–18 kg were housed individually in stainless steel cages in air-conditioned quarters and fed Purina monkey chow (Ralston Purina, St. Louis, MO) and fresh fruit and/or vegetables daily and water ad libitum. Females were paired with males for 5 d at the anticipated time of ovulation and pregnancy subsequently confirmed as described previously (21). Peripheral saphenous blood samples were obtained at 1- to 4-d intervals during the study period and from the maternal uterine vein at the time of placental-fetal delivery, and serum stored at –20 C until assayed for estradiol levels as described previously (22). Animals were cared for and used strictly in accordance with U.S. Department of Agriculture regulations and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (86-23, 1985). The Animal Care and Use Committee of Eastern Virginia Medical School approved the experimental protocol used in this study.

Tissue collection and preparation
Placentas were obtained by cesarean section from ketamine-sedated-isoflurane anesthetized baboons on d 100 (mid, n = 8) and 165 (late, n = 9) of gestation (term = d 184), the decidua basalis and chorionic plate removed, and sections of villous placenta placed in phosphate-buffered formalin (Sigma Chemical Co., St. Louis, MO) for subsequent immunocytochemical studies or in OCT mounting medium and stored at –80 C for isolation of specific cells by laser capture microdissection (LCM). Approximately 30–60 g of whole villous tissue was used for preparation of syncytiotrophoblast cytosolic and membrane fractions essentially as described by Kamath and Smith (23) and modified by us for use in baboon placenta (15). Briefly, villous tissue was minced in PBS (pH 7.4; Sigma), stirred with a magnetic stir bar for 60 min, filtered, and the filtrate and particulate fractions collected. The filtrate was differentially centrifuged to pellet debris and nuclei (800 x g), intracellular organelles, and vesicles, presumably including recycling endosomes associated with the cytoplasm and the juxtanuclear compartment (10,000 x g, i.e. 10K fraction) and a membrane pellet (150,000 x g) from which the MVM were collected. The particulate fraction was washed, sonicated to release intracellular organelles and vesicles including recycling endosomes in the cytoplasm and juxtanuclear compartment not released by mechanical disruption as well as membranes contiguous with the basal membrane (BMm), and then sonicated again to release the BM, which was obtained by differential centrifugation. Although we previously confirmed using the procedures outlined above (15, 16) that alkaline phosphatase activity was enriched more than 10-fold in the MVM and was not a major component of the 10K fraction or detected in other fractions and that binding of dihydroalprenolol (DHAP) was enriched 16-fold in the BM fraction, activity/binding of these factors was also determined in the MVM and BM of animals of the current study. Finally, because repeated sonication was required to remove the BM, this fraction, although enriched for BM, also contained components of the inner villous core including FVE. Therefore, this fraction is more appropriately termed the BM/FVE.

Immunocytochemistry
Sections (4 µm) of formalin-fixed, paraffin-embedded placentas or archived adult baboon kidney (8) were blocked for 30 min in 5% normal goat serum (Vector Laboratories, Burlingame, CA) and then incubated overnight at 4 C with primary antibodies to NHE1, NHE3, NHERF1, or NHERF2 diluted 1:125 to 1:7000 in 5% normal goat serum (NGS)-PBS essentially as described previously (8, 15) or with monoclonal antibody (Transduction Laboratories, Lexington, KY) to the endosomal protein marker, Rab4 (24), diluted 1:1000 in 5% NGS-PBS. After washing in PBS, sections were incubated for 2 h with biotinylated antirabbit or antimouse second antibody (Dako Corp., Carpinteria, CA) and then incubated for 30 min in the dark with streptavidin conjugated with AlexaFluor 488 Green (Molecular Probes Inc., Eugene, OR) diluted 1:500 with 5% NGS-PBS. After incubation for 10 min with Sudan Black (1% in 70% methanol; Sigma) to quench autofluorescence, slides were rinsed and treated with propidium iodide (1.0 µg/ml PBS) for 1 min to stain nuclei red. After application of mounting media [90% glycerol in 0.1 M Tris buffer (pH 9.0) containing 0.0625% n-propyl galate, final concentration], slides were sealed with nail polish and stored in the dark at 4 C until examined using a Zeiss 510 confocal laser microscope (Zeiss Inc., Heidelberg, Germany) or an Olympus BX41 microscope (Optical Elements Corp., Melville, NY) equipped with an Olympus DP70 digital camera with FITC, TRITC, and FITC/TRITC filter sets. Under these conditions, expressed proteins were detected as green and those colocalized with nuclei detected as yellow. To further evaluate localization of antiporters to syncytiotrophoblast nuclei, an additional series of placental sections were treated before incubation with primary antibodies with Image-iT FX signal enhancer (Molecular Probes) for 30 min, blocked in 5% NGS, and treated with streptavidin/biotin blocking reagent (Vector) according to the manufacturer’s instructions to block nonspecific binding to streptavidin receptors/binding proteins and endogenous biotin. Finally, studies were also performed with sections incubated without primary antibody or with primary antibody preabsorbed with immunizing peptide.

Western blot analyses
Western blots were performed essentially as described previously (8). Briefly, after determination of protein concentration by the bicinchoninic acid procedure (Sigma), syncytiotrophoblast fractions were loaded (15 µg protein/lane) onto discontinuous 8% (NHE1, NHE3) or 12% (NHERF1, NHERF2) sodium dodecyl sulfate (SDS)-polyacrylamide minigels, electrophoresed, wet transferred to Immobilon P (Millipore Corp., Bedford, MA), and membranes blocked with 3% fraction V BSA in 50 mm Tris (pH 7.5), containing 150 mm NaCl and 0.05% Tween 20 (Bio-Rad). Samples were incubated for 1 h at 30 C with polyclonal antibodies to the human proteins NHERF1 and NHERF2 generously provided by Dr. Chris Yun (Johns Hopkins University, Baltimore, MD) and commercial monoclonal and polyclonal antibodies, respectively, to NHE1 and NHE3 (Chemicon International, Temecula, CA). Primary antibodies were diluted to 1:10,000 (NHERF1, NHERF2), 1:400 (NHE1), or 1:200 (NHE3) in 50 mm Tris (pH 7.5), containing 150 mm NaCl, 0.05% Tween 20, 0.05% IGEPAL CA-630, and 1.5% BSA. After incubation with primary antibody, all membranes were then incubated with goat antirabbit or horse antimouse IgG horseradish peroxidase-conjugated second antibody (Vector) and proteins detected using enhanced chemiluminescence (Amersham). Specificity of the primary antibodies for analysis of NHEs and NHERFs was confirmed in previous studies (8). To compare the levels of antiporters or regulatory factors in the various fractions at mid- and late gestation, Western blots were developed in the same film cassette for the respective protein. After film development, samples were quantified by two-dimensional densitometry using an Image 1 analysis system (Universal Imaging Corp., Reamstown, PA) and results expressed as arbitrary densitometric units per microgram protein (15).

LCM of placental cells
Isolation of placental cells was performed by LCM using methods developed in our laboratories and described in detail previously (25). Briefly, serial 14-µm sections of placentas frozen in OCT and obtained at mid- and late gestation were mounted onto SuperFrost Plus glass slides (Fisher Scientific, Pittsburgh, PA), fixed in 75% ethanol, washed with distilled water, and then stained with HistoGene staining solution (Arcturus Engineering, Inc., Mountain View, CA). After washing in distilled water and incubation (30 sec) in 70, 95, and 100% ethanol, samples were incubated in xylene and air dried. An Arcturus PixCell II LCM system equipped with Olympus microscope (Arcturus) was used to capture onto LCM caps (Capture transfer film TF100, Arcturus) approximately 5 mm2 area of nonendothelial cells from the inner villous core. The LCM cap with captured cells was then tightly fitted to an Eppendorf tube to which was added 45 µl of 2x Tris-glycine SDS sample buffer (Invitrogen, San Jose, CA) containing 2.5% ß-mercaptoethanol (Sigma), 50% tissue protein extraction reagent (Pierce Biotechnology, Rockford, IL), and 1% protease inhibitor cocktail (Sigma). The cap was inverted, incubated at 75 C for 60 min, microcentrifuged, and lysates pooled into a single Eppendorf tube and stored at –80 C. Expression of NHERF2 was determined by Western blot as outlined above.

Na+/H+ exchange in MVM vesicles
MVM isolated from placentas at mid- (n = 5) and late (n = 5) gestation and stored (–80 C) in 50 mM Tris buffer (pH 6.9) containing 250 mM sucrose and not previously thawed were centrifuged at 15,000 x g for 30 min (4 C), washed three times with 500 µl ice-cold intravesicular buffer (IVB; pH 6.5) comprised of 25 mM 2[N-morpholino]ethenesulfonic acid (Sigma) and 150 mM KCl, and incubated for 30 min (37 C) in 200 µl IVB containing 10 µM 2',7'bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-acetoxymethyl ester (Molecular Probes). After washing with IVB, samples were incubated for 15 min (4 C) in 600 µl IVB or IVB containing 0.5 mM amiloride (Sigma). MVM vesicles (200 µg protein per 600 µl IVB) were then placed into one chamber of a KinTek SF 2001 Stopped Flow spectrofluorimeter (Kin-Tek Laboratories, La Marque, TX) and 2 ml of extravesicular buffer (EVB; pH 9.5) comprised of 10 mM HEPES (Sigma) and 150 mM NaCl added to a second chamber. After equilibration to 20 C, 60 µl MVM (20 µg protein) was mixed with 150 µl EVB (or IVB) and the voltage output (fluorescence) recorded for 30 sec. The pH of the reaction mixture determined in preliminary studies was 8.2 after addition of EVB. The automated computer-driven stopped-flow technology resulted in instantaneous mixing of MVM vesicles and EVB and continuous determination (six data points per second) of fluorescence (excitation 490 nm; emission 535 nm). At the conclusion of the 30-sec reaction, sample was displaced and another 60 µl MVM and 150 µl EVB were mixed and the analyses repeated. Thus, for each MVM sample, 10 determinations of NHE activity in presence/absence of amiloride were performed with the computer software determining a best-fit two exponential of the average of six of the analyses as well as amplitude (A2), adjusted for differences in loading, and rate constant (k2) from which t1/2 (k2 = 0.7/t1/2) and Na+/H+ activity (A2 x k2) expressed as millivolts per second per 20 µg protein were calculated. Preliminary studies confirmed that leakage of dye from loaded vesicles was minimal (<2%) over a 60-min period and that comparable amount of dye was released by treatment with 1% Triton X-100.

Statistics
Data are expressed as overall mean (± SE) and analyzed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum estradiol and placental and fetal growth
Maternal peripheral serum estradiol levels increased from approximately 1 ng/ml on d 85–120 of gestation to 2.5–3.5 ng/ml by d 165 (not shown), developmental changes that reflect placental production. Thus, mean (± SE) estradiol levels in uterine venous serum were 2-fold greater (P < 0.05) on d 165 (Table 1Go) than on d 100. Placental and fetal body weights also increased 2- and 4-fold, respectively, resulting in a 2-fold increase (P < 0.05) in the ratio of fetal to placental weight (Table 1Go).


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  		TABLE 1. Mean (± SE) placental and fetal body weights and uterine venous serum estradiol (E2) levels at mid- and late gestation in baboons

 
The activity of alkaline phosphatase in placental whole homogenate and MVM was approximately 3- to 4-fold greater (P < 0.05) at late than at midgestation (Table 2Go). Thus, alkaline phosphatase was comparably enriched approximately 11- to 18-fold in the MVM isolated from baboon placentas obtained at mid- and late gestation. In contrast, DHAP binding in placental homogenate was 2-fold lower (P < 0.05) and in BM/FVE 50% higher (P < 0.05) at late gestation, compared with respective values at midgestation. As a consequence, enrichment of DHAP in BM/FVE was greater (P < 0.05) in late than midgestation.


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  		TABLE 2. Alkaline phosphatase activity and DHAP binding in membrane fractions isolated from baboon placenta at mid- (n = 6) and late (n = 6) gestation

 
Of the trophoblast fractions examined, protein levels were greatest in the BMm (Table 3Go). The concentrations of protein in the MVM and BM/FVE fractions were approximately 60% greater (P < 0.05) in late than midgestation.


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  		TABLE 3. Mean ± SE concentration of soluble protein in cytosol and membrane fractions isolated from baboon placenta at mid- (n = 6) and late (n = 6) gestation

 
Expression of NHE3 and NHERF2
As assessed by confocal laser (Fig. 1Go, A and B) as well as immunofluorescent (Fig. 1Go, C–F) microscopy, NHE3 protein was abundantly expressed in the syncytiotrophoblast at midgestation (Fig. 1Go, A, C, and E) and detected as green in the cytoplasm, presumably associated with recycling endosomes, and in the juxtanuclear (i.e. perinuclear) compartment. The latter was confirmed by extensive expression of yellow dots indicative of colocalization with propidium iodide around syncytiotrophoblast nuclei but not the nucleolus (Fig. 1Go, C and E, inset 3). Presence of recycling endosomes in syncytiotrophoblast was confirmed by detection of the endosome-specific marker Rab4, which was extensively expressed in the cytoplasm but not the nucleus at midgestation (Fig. 2Go, A and C). Although NHE3 was lightly expressed in the BM and FVE (Fig. 1AGo), protein was not detected in the MVM. In contrast, in adult baboon kidney, NHE3 was abundantly expressed and detected in the MVM of the proximal tubules as well as in the cytoplasm (Fig. 1HGo). Antibody specificity was confirmed by absence of staining in sections incubated without primary antibody or with primary antibody preabsorbed with immunizing peptide (Fig. 1GGo). By late gestation, placental NHE3 expression (Fig. 1Go, B, D, and F) was markedly reduced in the juxtanuclear compartment (Fig. 1BGo), and thus, syncytiotrophoblast nuclei remained dark (Fig. 1Go, D and F, inset 1) and did not exhibit yellow dots that otherwise are indicative of colocalization with propidium iodide (Fig. 1Go, D and F, inset 3). However, NHE3 expression remained relatively abundant in the syncytiotrophoblast cytoplasm (Fig. 1Go, D and F, inset 1) and was still detectable in the FVE and lightly expressed in the BM (Fig. 1BGo).


Figure 1
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  		FIG. 1. Representative photomicrographs of the immunocytochemical expression of NHE3 in placental syncytiotrophoblast of baboons at d 100 (A, C, and E; n = 6) and 165 (B, D, and F; n = 6) of gestation (term = d 184) and in adult baboon kidney (H). All sections were incubated with polyclonal antibody to NHE3, stained with streptavidin conjugated with AlexaFluor 488, and except for the kidney treated with propidium iodide to stain nuclei red. Proteins localized to cytoplasm or membranes were detected by confocal (A and B) or fluorescence (C–H) microscopy as green and those colocalized to nuclei stained as yellow. Section in G was incubated without primary antibody. Original magnification, x400 (A, B, H) or x1000 (C–F). The insets (C, D, and H) were enlarged 2-fold. n, Nucleus; nl, nucleolus; cyto, cytoplasm.

 

Figure 2
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  		FIG. 2. Representative photomicrographs of the endosomal protein marker Rab4 at mid- (A–D) and late (E–H) gestation (n = 3 animals per group) as examined by fluorescent microscopy (original magnification, x400) as described in the legend to Fig. 1Go. Sections in D and H were incubated without primary antibody.

 
Rab4 expression and presumably recycling endosomes were also present in syncytiotrophoblast in late gestation (Fig. 2Go, E and G). Specificity was confirmed by absence of staining in sections incubated without primary antibody (Fig. 2Go, D and H). NHE3 was detected by Western blot (Fig. 3Go) as an 85-kDa protein in the BMm and 10K, cytoplasmic fractions that presumably contained vesicles (e.g. endosomes) but was not detected in the MVM. Specificity was further confirmed by absence of signal in samples incubated with primary antibody preabsorbed with immunizing peptide (Fig. 3CGo). Because the methods used in this study did not permit distinction between cytoplasmic vesicles/endosomes and proteins localized specifically to the juxtanuclear compartment, mean levels (arbitrary units x 10–3 per microgram protein) of NHE3 protein in the BMm (Fig. 3AGo) and 10K (Fig. 3BGo) fractions at midgestation (BMm, 33 ± 5; 10K, 52 ± 4) were similar to respective values at late gestation (BMm, 41 ± 5; 10K, 55 ± 3).


Figure 3
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  		FIG. 3. A and B, Representative Western immunoblot (top panels) and mean (±SE) concentrations (arbitrary units per microgram protein) of the 85-kDa NHE3 protein in the BMm (A) and 10K (B) fractions of syncytiotrophoblast prepared as outlined in Materials and Methods from baboon placentas obtained at mid- (d 100; n = 6) and late (d 165; n = 6) gestation. C, Comparison of NHE3 expression in BMm, 10K, and MVM fractions isolated from baboon placenta obtained at midgestation. Proteins were loaded (15 µg/lane) onto discontinuous SDS polyacrylamide gels, transferred to Immobilon P, and incubated with primary antibody to human NHE3. Block represents protein in BMm and 10K fractions probed with NHE3 antibody preabsorbed with immunizing peptide. Respective mean (±SE) values of NHE3 in the BMm (A) or 10K (B) fractions are not significantly different (P > 0.2; Student’s t test) at mid- and late gestation.

 
NHERF2 protein was abundantly expressed at midgestation (Fig. 4Go, A, D, and F) in the cytoplasm, presumably associated with endosomes as well as in the perinuclear compartment as confirmed by yellow color indicative of colocalization with propidium iodide around syncytiotrophoblast nuclei (Fig. 4Go, D and F, inset 3) but not in the nucleolus (Fig. 4DGo, inset 3). In contrast to NHE3, however, juxtanuclear expression of NHERF2 was not observed in all regions of the syncytiotrophoblast examined (Fig. 4DGo, insets 1 and 3). NHERF2 expression was abundant in the FVE (Fig. 4AGo), minimal in the BM and nonendothelial cells of the inner villous core, and minimal to nondetectable in the MVM (Fig. 4AGo). As with NHE3, by late gestation, expression of NHERF2 in the juxtanuclear compartment of all placental sections examined became reduced (Fig. 4Go, B and E, inset 1 and 3) but appeared to remain abundant in the syncytiotrophoblast cytoplasm (Fig. 4Go, B, E, and G, insets) and FVE (Fig. 4BGo) but minimal to nondetectable in the MVM and nonendothelial cells of the inner villous core. Specificity was confirmed by absence of staining in sections incubated without primary antibody (Fig. 4CGo). Western blot also showed that mean level (arbitrary units x 10–3 per microgram protein) of the 45-kDa NHERF2 protein, which was detected as a doublet in the BM/FVE fraction, was increased (P < 0.05) 3-fold between mid- (14 ± 2) and late (39 ± 5) gestation (Fig. 5AGo). The latter presumably reflected increased NHERF2 expression in the FVE. Thus, consistent with immunocytochemistry, NHERF2 protein expression determined by Western blot was minimal in nonendothelial cells isolated by LCM from the inner villous core at both mid- and late gestation (Fig. 5BGo).


Figure 4
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  		FIG. 4. Representative photomicrographs of NHERF2 expression in baboon placental syncytiotrophoblast at d 100 (A, D, and F; n = 4) and 165 (B, E, and G; n = 4) of gestation. Sections incubated without (C) or with polyclonal antibody to NHERF2, stained with AlexaFluor 488 and propidium iodide, and examined by confocal (A and B) or fluorescent microscopy (C–G) as described in legend to Fig. 1Go. Original magnification, x400 (A and B) or x1000 (D–G); insets (D and E) enlarged 2-fold. n, Nucleus; nl, nucleolus; cyto, cytoplasm.

 

Figure 5
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  		FIG. 5. A, Representative Western immunoblot and mean (±SE) concentrations (arbitrary units per microgram protein) of the 45-kDa NHERF2 protein in syncytiotrophoblast BM/FVE isolated from placentas obtained at mid- (d 100; n = 6) and late (d 165; n = 6) gestation and analyzed as described in legend to Fig. 3Go. The asterisk indicates values different from each other at P < 0.05 (Student’s t test). B, Western immunoblot of NHERF2 expression in the BM/FVE fraction and nonendothelial cells isolated by laser capture microdissection from baboon placentas at mid- and late gestation.

 
Expression of NHE1 and NHERF1
NHE1 was abundantly and comparably expressed in syncytiotrophoblast MVM at both mid- (Fig. 6AGo) and late (Fig. 6BGo) gestation. In contrast, NHE1 was lightly expressed in the BM at midgestation (Fig. 6AGo), and although expression at this site appeared to be increased in late gestation in some villi (Fig. 6BGo), this increase was not uniformly observed. At both mid- and late gestation, however, NHE1 expression was minimal in the cytoplasm, lightly detected in FVE, and negligible around syncytiotrophoblast nuclei. Specificity was confirmed by absence of staining in sections incubated without primary antibody (Fig. 6CGo). As seen in Fig. 6DGo, NHE1 was detected by Western blot as a 104-kDa protein, and consistent with immunocytochemistry, levels (arbitrary units x 10–3 per microgram protein) in the MVM and BM/FVE (not shown) were not significantly altered with development.


Figure 6
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  		FIG. 6. Representative photomicrographs of the immunocytochemical expression of NHE1 in baboon placental syncytiotrophoblast at d 100 (A, n = 4) and 165 (B, n = 4) of gestation. Original magnification, x400. C, Section of midgestation placenta incubated without primary antibody. D, Representative Western immunoblot and mean (±SE) concentrations of the 104-kDa NHE1 protein in syncytiotrophoblast MVM isolated from placentas obtained at mid- (d 100; n = 6) and late (d 165; n = 6) gestation.

 
NHERF1 was abundantly expressed in syncytiotrophoblast MVM and the FVE at mid- and late gestation but was not detected around nuclei and only weakly expressed in the cytoplasm and BM (Fig. 7Go, A and B). Immunocytochemical expression of NHERF1 did not appear to be affected by development. This was confirmed by Western blot (Fig. 7DGo), which showed that levels of the 50-kDa NHERF1 protein in the MVM were similar at mid- (50 ± 3) and late gestation (44 ± 7). Specificity was confirmed by absence of staining in sections incubated without primary antibody (Fig. 7CGo).


Figure 7
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  		FIG. 7. Representative photomicrographs of the immunocytochemical expression of NHERF1 in baboon placental syncytiotrophoblast at d 100 (A, n = 4) and 165 (B, n = 4) of gestation. C, Section of midgestation placenta incubated without primary antibody. Original magnification, x400. D, Representative Western immunoblot and mean (±SE) specific concentrations of the 50-kDa NHERF1 protein in syncytiotrophoblast MVM isolated from placentas obtained at mid- (d 100; n = 6) and late (d 165; n = 6) gestation.

 
Figure 8Go depicts representative time course of Na+/H+ exchange activity of MVM vesicles prepared from baboon placenta at mid- (A) and late (B) gestation. The original and computer-generated two-exponential fits of the average of six experimental determinations of change in fluorescence (i.e. voltage) of BCECF-loaded MVM vesicles incubated for 30 sec with buffer, which raised pH from 6.9 to 8.2 and extravesicular NaCl from 0 to 150 mM, are depicted. After a rapid (t1/2 < 2 sec) initial period of exchange, Na+/H+ exchange increased relatively more slowly (t1/2 = 15–17 sec) and began to plateau thereafter. The latter Na-dependent component of Na+/H+ exchange (A2, k2) reflected coupled activity of an NHE antiporter in the MVM and not uncoupled H+ or Na+ fluxes was confirmed by studies showing that after initial equilibration, Na+/H+ exchange (i.e. change in fluorescence) was inhibited more than 98% in vesicles incubated with 0.5 mM amiloride (not shown). Moreover, fluorescence did not change when BCECF-loaded vesicles were incubated with buffer to which NaCl was omitted (not shown). As summarized in Fig. 8CGo, mean Na+/H+ exchange activity (3.51 ± 0.39 mV) and t1/2 (18.0 ± 1.0 sec) of Na+-dependent Na+/H+ exchange exhibited by MVM vesicles at midgestation were similar to respective values (3.30 ± 0.55 mV; 16.4 ± 1.1 sec) in MVM vesicles in late gestation.


Figure 8
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  		FIG. 8. A and B, Representative time course of Na+/H+ exchange activity of MVM vesicles prepared from baboon placentas at mid- (A) and late (B) gestation. Exponential fit (Y = A1(–k1t) + A2(–k2t) + C) of change in fluorescence (i.e. voltage) of BCECF-loaded MVM vesicles incubated for 30 sec with buffer, which raised extravesicular NaCl to 150 mM. A1, k1, Initial period of exchange; A2, k2, Na+-dependent Na+/H+ exchange. C, Overall mean (±SE) Na+/H+ exchange activity (millivolts per second per 20 µg protein) of MVM vesicles isolated from baboon placentas at mid- (n = 5) and late (n = 5) gestation and calculated as the product of the rate constant and amplitude of the second exponential (k2 and A2). t1/2, 0.7/k2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the current study are the first to demonstrate that the subcellular distribution and levels of key components of the Na+/H+ exchange system in the baboon syncytiotrophoblast were altered between mid- and late gestation. Thus, the abundant expression of NHE3 and NHERF2 detected at midgestation associated/within the juxtanuclear compartment was markedly decreased near term. Although expression of NHE3 and NHERF2 in other regions (e.g. cytoplasm) of the syncytiotrophoblast did not change with development, levels of NHERF2 in the BM/FVE, as assessed by Western immunoblot, exhibited a developmental increase. In contrast, the levels of NHE1 and NHERF1 in the MVM, their primary site of expression, were not significantly altered with advancing gestation despite an increase in the concentration and thus amount of total proteins as well as the specific activity of alkaline phosphate associated with the MVM. Moreover, Na-dependent Na+/H+ exchange activity, which was inhibited by amiloride and thus attributable to an NHE antiporter (i.e. NHE1), was measurable in MVM and similar at mid- and late gestation. These findings link NHE1 protein expression and function.

The factors regulating the developmental changes in expression of NHE3 and NHERF2 remain to be determined. It is well established, however, that transcriptional activation of the NHE3 gene (20) and NHE3 expression and activity in the kidney (19) are up-regulated by cortisol. Although trafficking of NHE3 to/from recycling endosomes from/to the cell membrane has been shown to occur in Chinese hamster ovary cells via a phosphatidylinositol 3-kinase-dependent pathway (26), the factors(s) activating this pathway remain to be determined. We previously showed that estrogen, the levels of which increase between mid- and late gestation in the primate (27), regulated both the developmental increase in the levels and change in localization of the 11ß-HSD1 and 2 proteins within the placental syncytiotrophoblast (15, 16), biochemical/structural events that we proposed underpinned the marked increase in placental metabolism of cortisol to cortisone with advancing gestation (for review see Ref.18). Moreover, we showed that the 11ßHSD2 enzyme catalyzing the conversion of cortisol to cortisone was also juxtaposed to the syncytiotrophoblast nuclei, although we did not determine whether expression at this site was estrogen dependent (16). Therefore, the decline in syncytiotrophoblast NHE3 and NHERF2 expression in the juxtanuclear compartment of the primate placental syncytiotrophoblast with advancing gestation in animals of the current study is associated with an estrogen-dependent increase in placental removal of cortisol.

It is possible therefore that estrogen regulates NHE3 and NHERF2 levels in the juxtanuclear compartment of the primate placenta directly and/or indirectly by controlling local levels of cortisol, although the latter remains to be determined. Whether the developmental increase in levels of NHERF2 in the BM/FVE is an estrogen-dependent event and/or simply reflects the marked increase in the number/density of blood vessels per unit area of villous placenta with advancing gestation (28) also remains to be determined. Nevertheless, the baboon provides an excellent experimental model to ascertain in vivo regulation of the developmental expression of the Na+/H+ exchange system in the placenta.

Based on studies in proximal tubule-derived opossum kidney and other immortalized cell lines, the majority (>85%) of NHE3 protein is intracellular and localized to recycling endosomes (10, 24, 29) in the cytosol and associated with the juxtanuclear compartment (9). Moreover, it appears that these intracellular sites serve as reservoirs from which NHE3 recycles to and from the plasma membrane (10). The results of the current study showed that baboon placental syncytiotrophoblast also expressed the endosome protein maker Rab4 at both mid- and late gestation. Thus, it is possible, but remains to be confirmed, that NHE3 and perhaps NHERF2 expression in the baboon placenta are also largely intracellular and that these proteins may be associated with recycling endosomes in the cytoplasm and perhaps the juxtanuclear compartment. However, additional experiments are required to establish the specific vesicular compartments in which NHE3 and NHERF2 reside within the baboon placental syncytiotrophoblast. Interestingly, NHERF2 as well as NHERF1 have been implicated in the targeting and trafficking of the cystic fibrosis transmembrane regulator (30) and the ß2-adrenergic receptor (31). However, the movement or trafficking of proteins and the specific vesicular compartments involved in trafficking is highly complex. Therefore, although NHERF2 was detected in the juxtanuclear compartment of the baboon placenta, additional studies are required to determine the vesicular compartments involved and whether NHERF2 plays a role in protein trafficking in the primate placental syncytiotrophoblast.

The potential role of NHE3 in recycling endosomes has been examined using in vitro approaches (9, 24). For example, in NHE3-transfected antiporter-deficient Chinese hamster ovary cells and opossum kidney proximal tubule-derived cells (24), it appears that endosomal NHE3 creates acidic microdomains in the epithelium important for trafficking of various proteins. Because the directionality of Na+/H+ transport for plasma membrane and intracellular NHE3 is determined by the gradients of these ions across the membrane (24), creation of acidic microdomains within the apical cytoplasm of the plasma membrane would facilitate absorption of nutrients and Na+ while maintaining intracellular pH. Interestingly, inhibition of endosomal NHE3 in opossum kidney cells not only alkalinizes endosomes but also reduces the fusion of these vesicles and thus apical receptor-mediated albumin endocytosis (32, 33). Whether NHE3 and/or NHERF2 protein, which we assume may be associated with placental syncytiotrophoblast juxtanuclear and/or cytoplasmic endosomes, play a role in Na+/H+ exchange at these sites and also serve to acidify the MVM and/or BM in the baboon placenta remains to be determined. Also, although NHERF2 has been shown to modulate the activity of NHE3 at the apical membrane in the kidney in part by linking to ezrin (34, 35), a role for this regulatory factor on juxtanuclear NHE3 activity in baboon syncytiotrophoblast remains to be determined.

In contrast to the developmental decrease in NHERF2 expression in the juxtanuclear compartment, the protein levels of this regulatory factor were increased in the FVE between mid- and late gestation. The reason(s) for this apparent differential regulation of NHERF2 in placenta and fetal vascular cells remains to be determined. However, differential regulation could reflect differences in local levels of cortisol, which decrease in the placenta with advancing gestation but increase in the fetus as a result of onset of cortisol production by the fetal adrenal gland (for review see Ref.36). Nevertheless, the increase in NHERF2 expression with advancing gestation in fetal vascular tissue coupled with expression of NHE3 and NHE1 at this site may reflect a switch in the ability of the fetus to initiate control of Na+/H+ exchange independently and/or in concert with the placenta in late gestation.

It is generally accepted that NHE1 serves housekeeping functions including maintenance of cell volume and intracellular pH (5, 37). In baboons of the current study, the abundant expression of NHE1 and its regulatory factor NHERF1 in the MVM as well as Na+/H+ exchange activity was maintained between mid- and late gestation despite a 4-fold increase in alkaline phosphatase activity and a concomitant 60% increase in the concentration of proteins associated with the MVM. NHE1 expression and Na/H exchange activity in human placental MVM has also been shown to be similar in the second and third trimester (38, 39). The maintenance of NHE1 and NHERF1 levels per unit of MVM may be physiologically significant and important to ensure continued movement of H+ ion from the placenta to the mother during the second half of gestation, a period when placental growth and presumably placental metabolic activity and generation of H+ ions are increased. Because H+ is a major regulator of NHE1 gene transcription in other epithelial cells, e.g. the kidney (40), expression of this antiporter in the primate placenta may also be sensitive to and thus regulated by H+ ion. However, in renal cells NHE1 is localized primarily to the basolateral membrane and not the MVM. Therefore, because the level of this antiporter in the placenta is localized to and thus more related to the level of MVM, factors controlling MVM development may play a more significant role in the extent to which NHE1 is expressed at this site.

Results of the current study support this suggestion. It is well established that microvilli are composed of core actin filaments and several actin-binding proteins including ezrin (41, 42, 43), which we showed was present in syncytiotrophoblast MVM (8). Ezrin binds protein kinase A and links F-actin filaments together to create a scaffold-like structure that serves as the frame for the formation of microvilli (41). Importantly, creation of the scaffold is dependent on the presence of NHERF1, originally termed ezrin binding protein 50 (44), which binds ezrin to anchor ezrin-actin to the plasma membrane via several plasma membrane proteins including NHE1 or NHE3 to complete formation of the microvilli (34). Thus, formation of MVM and syncytiotrophoblast Na+/H+ exchange may be linked, although the latter remains to be confirmed.

In summary, the results of the current study show that the subcellular distribution of key components of the Na+/H+ exchange system in baboon syncytiotrophoblast were markedly altered during gestation. Thus, expression of NHE3 and NHERF2 in the juxtanuclear compartment was decreased between mid- and late gestation. In contrast, NHERF2 expression in the fetal vessels was increased, perhaps reflecting transfer of control of Na+/H+ regulation at the basal side of the placenta to the fetus. Finally, because these developmental changes in antiporter expression were associated with the increase in estrogen production and the estrogen-dependent increase in placental metabolism of cortisol, it is possible but remains to be determined that estrogen may play a role in regulating the localization of the Na+/H+ exchange system in the primate placenta.


   Acknowledgments
 
We sincerely appreciate the secretarial assistance of Ms. Sandra Huband with the manuscript and preparation of the photomicrographs; R. B. Billiar, Ph.D., with advice and assistance with the Western blot analyses; and Howard White, Ph.D., with analysis of Na/H exchange activity using stopped-flow spectrofluorimeter. The generous supply of antibodies to NHERF1 and NHERF2 provided by Dr. Chris Yun (Johns Hopkins University, Baltimore, MD) is greatly appreciated.


   Footnotes
 
This work was supported by National Institutes of Health Research Grant R01 HD-13294.

The authors have nothing to disclose.

First Published Online March 9, 2006

Abbreviations: A2, Amplitude; BCECF, 2',7'bis(carboxyethyl)-5(6 )-carboxyfluorescein; BM, basal membrane; BMm, membrane contiguous with the basal membrane; DHAP, dihydroalprenolol; EVB, extravesicular buffer; FVE, fetal vascular endothelium; 11ß-HSD, 11ß-hydroxysteroid dehydrogenase; k2, rate constant; LCM, laser capture microdissection; MVM, microvillus membrane; NGS, normal goat serum; NHE, Na+/H+ exchanger; NHERF, NHE regulatory factor; SDS, sodium dodecyl sulfate.

Received July 14, 2005.

Accepted for publication March 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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