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Female Reproductive Tract |
a Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong
b Shanghai Institute of Planned Parenthood Research, Shanghai, China
c Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Na+/H+ exchanger (NHE) is a ubiquitous plasma membrane transporter in mammalian cells. Six NHE isoforms have been identified, four of which are expressed in epithelial cells. NHE1 is considered to ubiquitously exist in almost all mammalian cells [9] and to play a housekeeping role in regulating intracellular pH (pHi) [10], osmolarity, and cell volume. NHE1 was believed to be present in the epithelial cells exclusively in the basolateral membrane [11] until the recent demonstration of its bilateral localization in the kidney, jejunum, and colon [12]. Both NHE2 and NHE3 are specifically expressed in the apical membrane of epithelial tissues such as the kidney [13, 14], intestine [15], and epididymis [16, 17]. They are believed to be responsible for luminal Na+ reabsorption [18, 19]. However, in a cell line of the inner medullary collecting duct, NHE2 coexist with NHE1 on the basolateral membrane [20], where they may serve similar functions. NHE4 is localized to the basolateral membrane of the stomach and kidney [21], but little is known about its physiological functions.
It appears possible that NHEs may also be expressed in the endometrial epithelium and may be responsible for H+ extrusion in order to sustain HCO3- secretion across the endometrium, as occurs in pancreatic duct HCO3- secretion [22]. However, the distribution and function of NHEs in endometrial epithelial cells have not been investigated to any extent. We undertook the present study to investigate the mRNA expression and immunolocalization of different NHE isoforms as well as their functional contribution to H+ extrusion from cultured mouse endometrial epithelium. The results demonstrated expression of multiple NHEs in mouse endometrium, which may play different roles in uterine functions depending on their cellular locations, namely the apical and basolateral membranes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco modified Eagle medium with nutrient mixture F-12 (DMEM/F-12), PBS, fetal bovine serum (FBS), nonessential amino acids, pancreatin, a reverse transcriptase-polymerase chain reaction (RT-PCR) kit and NBC primers were purchased from Gibco Invitrogen (Grand Island, NY). Penicillin/streptomycin, trypsin (type II), forskolin, N-methyl-D-glucamine (NMG), Hepes, polyoxyethylene-sorbitan monolaurate (Tween-20), BSA, and nigericin were obtained from Sigma Chemical Company (St. Louis, MO). Amiloride hydrochloride was obtained from Research Biochemicals International (Natick, MA). 2',7-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, OR). Millipore filters and Matrigel were purchased from Collaborative Biochemical Products (Bedford, MA). Triton X-100 was from Boehringer-Mannheim GmbH (Mannheim, Germany).
Cell Isolation and Culture
Endometrial epithelial cells were enzymatically isolated from the mouse uterus according to the method described by McCormack and Glasser [23] with slight modifications [24]. The use of animals in the present project was authorized by the Animal Research Ethics Committee of The Chinese University of Hong Kong. Uteri were obtained from 3.5- to 4-wk-old immature ICR mice to avoid the complication of the estrous cycle. On average, the uteri taken from 10 mice were used to obtain enough endometrial epithelial cells for making 12 filters of cultures. Uteri were washed in sterile PBS (without Ca2+ and Mg2+). After trimming off the fatty and connective tissues, the uteri were sliced longitudinally. All sliced uteri were put together into a 15-ml tube containing 10 ml PBS supplemented with 6.5 mg/ml trypsin and 25 mg/ml pancreatin. The tube was kept at 0°C for 60 min and at room temperature for another 45 min. As the enzyme-containing PBS was carefully poured away, DMEM/F-12 culture medium containing 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids, 100 IU/ml penicillin, and 10 µg/ml streptomycin was added to stop the activity of the trypsin. The medium was replaced with PBS 5 min afterward. The tissues were then gently shaken for 30 sec. Uterine tissues were removed and the filtrate was centrifuged at 1000 x g for 5 min. Then the cell pellet was resuspended in DMEM/F-12 medium. The isolated endometrial cells were plated at a density of about 2–3 x 106 cells/ml onto floating permeable supports made of Transwell-Col membranes (Costar, U.K.) and a plastic ring (with cell growth area of 0.1 cm2). Each membrane was coated with 40 µl of diluted Matrigel (1:8 in PBS) and dried before cell plating. The cultures were supported by a small, folded glass rod in medium and incubated at 37°C in 95% O2/5% CO2 for 3 days until the cells formed a confluent, polarized monolayer before they were used for pHi measurements. This set of experiments was repeated more than six times.
RNA Isolation and RT-PCR
Total RNA was isolated from uteri of three immature mice using TRIzol reagent (Gibco Invitrogen). Ten micrograms of total RNA were used for first-strand cDNA synthesis using random hexamer primers and Superscript II RNase H-reverse transcriptase (Superscript Preamplification System; Gibco Invitrogen). The resulting first-strand cDNA was directly used for PCR amplification.
Different sets of primers of NHE isoforms were used for PCR analysis as described by Borensztein et al. [25]. The primers were constructed on the basis of the published cDNA sequence of rat NHE isoforms from GenBank. The two primers used for amplifying NHE1 (accession number M85299) were sense, 5'-TCTGCCGTCTCAACTGTCTCTA-3' and antisense, 5'-CCCTTCAACTCCTCATTCACCA-3', which generated a 422-base pair (bp) NHE1 PCR product. The two primers for NHE2 (L11004) were sense, 5'-GCAGATGGTAATAGCAGCGA-3' and antisense, 5'-CCTTGGTGGGGGCTTGGGTG-3', which generated a 310-bp NHE2 product. The two primers for NHE3 (M85300) were sense, 5'-GGAACAGAGGCGGAGGAGCAT-3' and antisense, 5'-GAAGTTGTGTGCCAGATTCTC-3', which yielded a PCR product of 321 bp. The two primers for amplifying NHE4 (M85301) were sense, 5'-GGCTGGGATTGAAGATGTATGT-3' and antisense, 5'-GCTGGCTGAGGATTGCTGTAA-3', which yielded a PCR product of 501 bp. The conditions for PCR reactions were as follows: 4 min at 94°C (initial melt); 30 1-min cycles at 94°C, 1 min at 60°C, 1.5 min at 72°C, and then 72°C for 15 min (final extension). For the negative control, RT-PCR was performed in the absence of RT. The PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide. The PCR products of the expected sizes were confirmed by sequencing. RT-PCR analysis of NHE isoforms in uterus was repeated more than three times.
To further confirm the finding, the RT-PCR experiments working with RNA obtained from cultured mouse endometrial epithelium were performed. The isolated cells were plated onto large filters (diameter 40 mm) and cultured under the same condition as described above. The confluent monolayer was scraped and collected to extract total RNA for RT-PCR.
Immunohistochemistry
Two sets of primary antibodies were tested via immunohistochemistry. One set of NHE1, NHE2, and NHE3 antibodies were raised in rabbit as previously described [15, 26]. The other set of NHE1–NHE4 antibodies were purchased from Chemicon, Inc. (Temecula, CA).
The uteri obtained from eight ICR immature mice were carefully excised and transferred to Bouins fixing solution and embedded in paraffin. Paraffin sections of 5 µm were dewaxed and hydrated. Antigens were retrieved by treatment in 0.01 M citrate buffer (pH 6.0) for 5 min in a microwave oven. They were then rinsed twice with pure water and incubated in methanol containing 3% (v/v) H2O2 for 15 min. After rinsing with pure water and PBS, sections were incubated in normal blocking serum (Vectastain Elite ABC kit, Vector PK-6101; Vector Laboratories, Burlingame, CA) for 30 min and then with the polyclonal rabbit anti-mouse NHE1, NHE2, and NHE4 antibodies diluted 1:500 (v/v) with diluting buffer (PBS with 0.01% v/v Triton X-100, 0.01% v/v Tween-20, and 0.1% w/v BSA) at 4°C overnight. Sections were then washed three times with PBS and incubated with biotinylated secondary antibody (avidin-biotin complex ;obABC;cb kit) for 30 min. After washing three times with PBS, the sections were incubated with Vectastain Elite ABC reagent (ABC kit) for 30 min and finally washed three times with PBS again. Reactivity was visualized by immersing sections in a peroxidase-substrate solution (diaminobenzidine ;obDAB;cb or a VIP substrate kit; Vector Laboratories) until the desired stain intensity developed. Slides were gently rinsed with pure water for 5 min, counterstained with Lillie-Mayer hematoxylin (Merck, Darmstadt, Germany), dehydrated, and mounted for observation. Negative controls were obtained by omitting primary antibodies. Kidneys obtained from ICR mice were used as positive controls. A semiquantitative assessment of the immunostaining for each NHE isoform was made. The scale 0 stands for no staining and 4 for most intense staining.
Measurement of NHE Activity
After confluency was achieved, a polarized monolayer was rinsed slightly with NaCl Ringer solution and loaded with BCECF-AM (3 µM), a pH-sensitive fluorescent dye, in NaCl Ringer solution for 30 min at 37°C. The sample was then mounted into a miniature Ussing chamber attached to the stage of an inverted microscope (TE300; Nikon, Tokyo, Japan). The chamber was constructed so that solutions bathing apical and basolateral surfaces could be changed rapidly and independently. The intracellular pH (pHi) was measured by the PTI Ratio-Master fluorescence system (Photon Technology International, Lawrenceville, NJ). The loaded dye in cells was alternately excited at two wavelengths (440 and 490 nm) and the emission was measured at wavelength 510 nm by a photometer. The ratio of the emitted light at an excitation of 490 nm to that at 440 nm in response to changes of intracellular pH was measured. The cultured cells were initially superfused with NaCl Ringer solution and then replaced with basolateral NH4Cl-containing solution for alkaline loading. NMG-Cl (Na+-free) Ringer solution was then introduced to result in a large acidification of the cells. The NMG-Cl solution was subsequently replaced with NaCl Ringer solution to induce pHi recovery of cells. At the end of each experiment, the fluorescence ratio (490:440) was calibrated to pHi by exposing monolayers to KCl-calibration Ringer solution containing 15 µM nigericin (the artificial K+-H+ ionophore) at four or more different extracellular pH (pHo) values [27]. The NaCl Ringer solution contained 150.0 mM NaCl, 2.5 mM K2HPO4, 1.3 mM CaCl2, 1.3 mM MgCl2, 5 mM D-glucose, and 10 mM Hepes. For NMG-Cl Ringer solution, NaCl was replaced in equimolar concentrations with NMG-Cl. For the NH4Cl solution, 20 mM NH4Cl replaced NaCl in equimolar concentrations. All Ringer solutions were adjusted to pH 7.4 (37°C). The KCl calibration solution was prepared by replacing 130 mM NaCl mole to mole with KCl and then titrating to different pHo levels between 6.0 and 8.0 with HCl or KOH. The NHE activity, expressed as 
pH/min, was calculated from the slope of the initial 100–200 sec of Na+-dependent pHi recovery.
Statistical Analysis
Data are expressed as means ± SEM. Compositions between groups of data were made with the Student t-test, with P < 0.05 considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the expression of NHE isoforms in mouse endometrium, RT-PCR was performed using RNA extracted from mouse uterus. The PCR products obtained using primers for NHE1, NHE2, and NHE4 showed the expected molecular weights; however, no band was detected for NHE3 (Fig. 1A). RT-PCR was also performed with RNAs extracted from isolated and cultured endometrial epithelial cells, and similar results were obtained (Fig. 1B). The mouse kidney cDNA was used in the experiment as a positive control because all four NHE isoforms had been shown to be expressed in this tissue [14, 21]. PCR products from the kidney were similar to those obtained from the uterus, except for NHE3.
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Immunolocalization of NHEs
Immunoreactive NHE1, NHE2, and NHE4, obtained using commercially available antibodies and a DAB substrate kit, were detected on both the apical and basolateral domains of the endometrial luminal and glandular epithelia, with stronger immunoreactivities associated with the luminal epithelium for all three NHEs (Fig. 2). Whereas NHE1 and NHE2 immunoreactivities were evenly distributed in both apical and basolateral domains (Fig. 2, A1 and B1), NHE4 immunoreactivity was more prominently detected in the apical membrane (Fig. 2, C1). However, positive controls showed that NHE1, NHE2, and NHE4 isoforms were all localized to the basolateral domain (Fig. 2, A3, B3, and C3) as previously reported [11, 20, 21], suggesting that bilateral distribution of NHEs observed in the present study was specific for endometrial epithelium. Immunofluorescence assay was also performed to confirm localization of NHE1, NHE2, and NHE4 in mouse endometrial epithelium (Fig. 3). Although all NHEs immunofluorescent stainings were distributed bilaterally, NHE4 was found to be less polarized compared to that observed with immunostaining (DAB, see above). The intensity of immunoreactivity shown in Figure 2 and a semiquantitative assessment of the immunostaining for each NHE isoform are listed in Table 1, in which scale 0 stands for no staining and 4 indicates the most intense staining.
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Functional Activity of NHE
In order to examine the functional activity of NHE, we studied the Na+ dependence of pHi recovery from acid loading and the sensitivity of the H+ extrusion process to the NHE inhibitor, amiloride. Perfusion with NH4Cl followed by an NMG-Cl solution induced acidification in the endometrial cells. The high-K+/nigericin method was used for calibration. As shown in Figure 4, the changes in fluorescence ratio (490:440) in response to changes in pHo were plotted, and therefore, the fluorometric measurements could be translated into changes in pH.
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After acidification, little pHi recovery was observed when Na+ was absent from both apical and basolateral solutions. When Na+ was reintroduced in the basolateral perfusate, while keeping the apical solution free of Na+, a rapid recovery of pHi was observed from 6.47 ± 0.25, to a steady value of 7.66 ± 0.31 (n = 8) (Fig. 5A), indicating the dependence of pHi recovery on Na+. However, the Na+-dependent pHi recovery could be completely inhibited by the presence of amiloride (500 µM), an inhibitor of NHE (n = 8) (Fig. 5B), further indicating that the Na+-dependent H+ extrusion was most likely mediated by basolaterally located NHEs. The pHi recovery could also be induced by reintroducing Na+ to apical solution while keeping the basolateral solution free of Na+ (Fig. 6A); however, the pHi recovery rate with Na+ present in the apical compartment was only 0.07 ± 0.02 pH/min compared with 0.87 ± 0.07 pH/min (n = 7) (Fig. 6B) observed when Na+ was present in the basolateral compartment, indicating that the contribution of basolaterally located NHE(s) to the pHi recovery was 12.4 times more than that of apically located NHEs. In contrast to the basolateral response, the presence of amiloride (500 µM) in the apical solution did not completely inhibit the apical Na+-induced pHi recovery response (n = 5) (Fig. 7), suggesting that one or more NHE isoforms involved in the apical response were different from those involved in the basolateral response.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whereas our immunohistochemical studies suggest the presence of all three NHEs in mouse endometrium, the immunolocalization of some of these isoforms in this tissue are different from that previously reported in other tissues or cell types. NHE1, for instance, in the present study was also observed in the apical membrane in addition to the basolateral membrane where it has been immunolocalized in many other tissues [11, 28, 29]. The bilateral distribution of NHE1 in mouse endometrium seemed to contradict the exclusive localization of NHE1 in the basolateral membrane found in all tissues examined. In order to make sure of this, we repeated the experiments many times with antibodies both from the laboratory and commercial sources, as well as using different substrate kits for immunolocalization, and the same results were obtained indicating bilateral immunolocation of NHEs in the endometrium. However, when the same antibodies were used to immunolocalize NHE1 in mouse kidney, immunoreactivity was found only in the basolateral membrane, suggesting a different distribution of NHEs in the endometrium than that in the kidney. A recent report has also demonstrated an interesting tissue-dependent subcellular localization of NHE1 to both the apical and basolateral membranes in kidney cortex, jejunum, and colon [12]. Similarly, NHE2 and NHE4 were also found bilaterally localized in this study although they have been found unilaterally localized in other tissues (e.g., in the intestine and renal IMCD3 cells).
Although all three NHE isoforms were found to be bilaterally localized, results from fluorometric studies suggest that the response of NHEs to intracellular acidification and therefore their contribution to pHi recovery are markedly different depending on their localization. The rate of pHi recovery depending on apical Na+ was much slower than that depending on basolateral Na+. A number of explanations may account for this observation. First, basolaterally located NHE1, NHE2, and NHE4 may function differently from their apical counterparts such that the basolaterally located NHEs are more responsible for pH regulation as in the case of NHE1. Second, apical and basolateral NHEs are subject to different regulatory mechanisms. Whereas the basolateral ones are sensitive to pH changes, the apical ones may be regulated by other physiological conditions or regulators. Finally, it is possible that the NHE responsible for apical Na+-induced pH recovery was different from the one responsible for basolateral response, meaning that not all NHEs present in the same membrane are simultaneously involved in pH recovery. For instance, the basolateral Na+-induced pH recovery could involve only NHE1, whereas the apical ones may predominantly involve NHE2 or NHE4. This notion is supported by the present observation of a more prominent immunolocalization of NHE4 in the apical region. The difference in amiloride sensitivity between apical and basolateral pH responses also seems to support this notion. NHE1, which can be inhibited by amiloride, is well known for its role in pHi regulation and therefore its major contribution to basolateral Na+-induced pHi recovery may explain the complete inhibition of basolateral response by amiloride. On the other hand, the apical Na+-induced pHi recovery appeared to be less sensitive to amiloride (Fig. 7), suggesting that different NHEs are involved. However, without specific inhibitors for different NHE isoforms, we are not able to assess the contribution of each of the NHE isoforms in detail.
HCO3- has been demonstrated to be important for sperm capacitation and embryo development [3, 5, 6] and the elevated HCO3- content in the uterine fluid compared with that in plasma indicates active transport of HCO3- across the endometrium. Our previous study has demonstrated that mouse endometrial epithelium secretes HCO3- when stimulated by cAMP-evoking agonists, forskolin, or adrenalin [7, 8, 24]. The secretion of HCO3- requires cellular accumulation of HCO3- through basolateral Na+-HCO3- cotransporter [8]. On the other hand, previous studies have also indicated that carbonic anhydrase in the cells may catalyze the hydration of CO2 to carbonic acid, which is further dissociated into H+ and HCO3- [7, 8]. While HCO3- is secreted into the lumen through apical pathways such as HCO3-/Cl- exchanger and the cystic fibrosis transmembrane conductance regulator (unpublished data), it also requires that H+ be exported out of the cells across the basolateral membrane. The present demonstration that NHE1, NHE2, and NHE4 are localized in the basolateral membrane indicates that they are the most likely candidates involved in the H+ extrusion process, although the contribution of specific NHE isoforms remains to be elucidated. NHE has also been proposed to be involved in basolateral H+ extrusion for HCO3- secretion in the pancreatic duct [22].
The endometrial epithelium secretes HCO3-, and the basolateral NHE activity is geared to sustain apical secretion of HCO3-. This may explain why the basolateral Na+-induced pHi recovery was 12.4 times faster than the apical Na+-induced one. During the process of HCO3- secretion, the activity of apical NHEs should be kept at a minimal level because apical H+ secretion would have a counteracting effect. Thus, the present finding of higher basolateral NHE activity supports a role for basolaterally located NHEs in HCO3- secretion across the endometrium. The question remains regarding the functional role of apically located NHEs. As suggested in many other epithelia, apical NHEs may be involved in Na+ absorption, especially in tissues in which no other alternatives, such as epithelial Na+ channels (ENaC), are present. The epididymis, for example, reabsorbs more than 90% of the seminal fluid but for a long time researchers were puzzled by the absence of ENaC in the epididymal epithelium because ENaC was considered to be the major mechanism for Na+ reabsorption on which fluid reabosption depends. The recent findings of multiple NHEs localized in the epididymal epithelial apical membrane are consistent with the earlier study demonstrating amiloride sensitivity of fluid absorption [30], and indicating an important role for NHEs in epididymal Na+ reabsorption. In contrast to the epididymis, however, the endometrium does express EnaCs, which have been found largely responsible for Na+ absorption amid basal conditions [7, 24, 30]. This suggests that apical NHEs may play a secondary role in Na+ absorption in the endometrium. However, our recent study has also demonstrated estrus cycle-dependent expression of ENaCs: they are highly expressed during diestrus but are relatively low at other stages [31]. Although we have not examined in great detail the expression pattern of NHEs throughout the estrus cycle, we did observe similar immunoreactivity of all three NHEs at proestrus and diestrus (data not shown). Therefore, it is possible that apical NHEs may also play a complementary role in Na+ absorption during some estrus stages when ENaC expression is low.
In summary, the present study demonstrated the expression of NHE1, NHE2, and NHE4 isoforms in mouse endometrium. The bilateral distribution of the isoforms suggests they play different roles in regulating the uterine fluid environment. In addition to their role in homeostatic control of pHi of endometrial cells, apical NHEs may participate in Na+ absorption, while basolateral NHE is responsible for extruding H+ to the blood in order to sustain HCO3- secretion out into the uterine fluid on which sperm capacitation and embryo differentiation depend. Further studies are required to characterize and elucidate the functional role of each of the NHE isoforms identified.
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FOOTNOTES |
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2 Correspondence: Hsiao Chang Chan, Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong. FAX: 852 2603 5022; hsiaocchan{at}cuhk.edu.hk
Received: 17 March 2002.
First decision: 8 April 2002.
Accepted: 7 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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