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Involvement of Na⁺-HCO₃^- Cotransporter in Mediating Cyclic Adenosine 3',5'-Monophosphate-Dependent HCO₃^- Secretion by Mouse Endometrial Epithelium¹

^a Epithelial Cell Biology Research Center ^b Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong ^c Shanghai Institute of Planned Parenthood Research, Shanghai, China

	ABSTRACT

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The present study investigated the involvement of Na⁺-HCO₃^- cotransporter in mediating cAMP-stimulated HCO₃^- secretion across the cultured mouse endometrial epithelium using the short-circuit current (I_SC) technique and intracellular pH measurement. Forskolin stimulated a rise in the I_SC, 55.6% and 52.1% of which could be reduced by the removal of extracellular Cl^- or by eliminating the contribution of Cl^- secretion by bumetanide, an inhibitor of Na⁺-K⁺-2Cl^- cotransporter, respectively. More than 80% reduction in the forskolin-induced I_SC was obtained when both Cl^- and HCO₃^- in the bath were removed or in HCO₃^--free solution with bumetanide, indicating that the I_SC depended on both Cl^- and HCO₃^-. The presence of the Na⁺ channel-blocker amiloride in the apical solution did not reduce the forskolin-induced I_SC; however, the I_SC could be abolished by removing Na⁺ from the bathing solution, suggesting that the Cl^-- and HCO₃^--dependent I_SC was also dependent on basolateral Na⁺. The forskolin-stimulated I_SC could be reduced 43.6% by removal of HCO₃^- and 47.9% by a Na⁺-HCO₃^--cotransporter inhibitor, dihydrogen-4,4'-didsothiocyanostilbene-2,2'-disulfonic acid (H₂DIDS). The inhibitory effect of H₂DIDS was observed in Cl^--free solution, but not when HCO₃^- was removed, thus confirming its effect on HCO₃^--dependent transport. Intracellular pH measurements demonstrated that the recovery from cellular acidification depended on the presence of both basolateral Na⁺ and HCO₃^-, further indicating the involvement of Na⁺-HCO₃^- cotransporter. Reverse transcription-polymerase chain reaction experiments confirmed the expression of Na⁺-HCO₃^- cotransporter in the mouse endometrium. The results suggest that basolaterally located Na⁺-HCO₃^- cotransporter is involved in mediating cAMP-stimulated HCO₃^- secretion across the mouse endometrial epithelium.

female reproductive tract, uterus

	INTRODUCTION

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Endometrial epithelium is the functional mucosal lining of the uterus, which is largely responsible for formation of the uterine fluid. Examinations of human uterine fluid composition have indicated active ion transport across the endometrial epithelium through absorptive and secretory activities [1]. Changes in the ionic composition of the uterine fluid, including those of HCO₃^- and Cl^-, have been observed during various reproductive events [2–4]. The HCO₃^- concentration in the uterine fluid has been reported to be from 2- to 4-fold higher than that in the plasma in a number of species [5], suggesting the involvement of an active HCO₃^- transport mechanism. Although the observation of high HCO₃^- content in the uterine fluid is more than half a century old and HCO₃^- is considered to be essential in certain reproductive processes, such as sperm capacitation and embryo development [6–8], the cellular mechanisms underlying the formation of HCO₃^--rich uterine fluid remain largely unknown.

Vishwakarma [5] has long speculated that a Na⁺-dependent mechanism is involved in active transport of HCO₃^- across the endometrial epithelium. Various mechanisms for HCO₃^- transport have been recently proposed to be involved in HCO₃^- secretion/absorption and intracellular pH (pH_i) regulation in a number of tissues, one of which involves basolaterally located Na⁺-HCO₃^- cotransporter (NBC). Functional NBC was first identified in the salamander (Ambystoma tigritum) kidney [9], then in the pancreas [10–15], colon [16], liver [17–19], heart [20, 21], and parietal cells [22].

Our previous studies have demonstrated that primary-cultured mouse endometrial epithelium exhibited a basal short-circuit current (I_SC) predominantly mediated by Na⁺ absorption [23]. On the other hand, I_SC response induced by cAMP-evoking agents, including adrenaline and prostaglandin, was predominantly contributed by Cl^- and HCO₃^- secretion [24, 25]. The Cl^- secretion has been shown to involve the basolateral Na⁺-K⁺-ATPase, Na⁺-K⁺-2Cl^- cotransporter, K⁺ channel, and apical Cl^- channels, most likely the cystic fibrosis transmembrane conductance regulator (CFTR) [26–28]. However, the mechanisms underlying HCO₃^- transport across the endometrial epithelium have not been adequately studied.

In the present study, we investigated the possible involvement of NBC in mediating HCO₃^- transport across cultured mouse endometrial epithelium using the I_SC technique, pH_i measurement, and reverse transcription-polymerase chain reaction (RT-PCR) method. Our results suggest that the basolaterally located NBC may play an important role in mediating cAMP-activated HCO₃^- secretion by mouse endometrial epithelium.

	MATERIALS AND METHODS

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Materials

Dulbecco modified Eagle medium with nutrient mixture F-12 (D-MEM/F-12), PBS, fetal bovine serum (FBS), nonessential amino acids, pancreatin, RT-PCR kit, and NBC primers were purchased from Gibco Invitrogen (Grand Island, NY). Penicillin/streptomycin and trypsin (type II), forskolin, N-methyl-2-glucamine (NMDG), and bumetanide were obtained from Sigma Chemical Co. (St. Louis, MO). Amiloride hydrochloride was obtained from Research Biochemicals International (Natick, MA). Dihydrogen-4,4'-didsothiocyanostilbene-2,2'-disulfonic acid (H₂DIDS) and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) were purchased from Molecular Probes (Eugene, OR). Millipore filters and Matrigel were purchased from Collaborative Biochemical Products (Bedford, MA).

Cell Isolation and Culture

Endometrial epithelial cells were enzymatically isolated from the mouse uterus according to the method described by McCormac and Glasser [29] with slight modifications [23]. Uteri were obtained from 3.5- to 4-wk-old, immature ICR mice to avoid the complication of the estrous cycle. Uteri obtained were washed in sterile PBS (without Ca²⁺ and Mg²⁺). After trimming off the fatty and connective tissues, the uteri were sliced longitudinally. The sliced uteri were then treated in PBS supplemented with 6.5 mg/ml of trypsin and 25 mg/ml of pancreatin at 0°C for 60 min and at room temperature for another 45 min. As the enzyme containing PBS was poured away carefully, D-MEM/F12 culture medium containing 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids, 100 IU/ml of penicillin, and 10 µg/ml of streptomycin was added to stop the activity of the trypsin. The medium was replaced with PBS 5 min later, and the tissues were then gently shaken for 30 sec. Uterine tissues were removed, and the filtrate was centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 12 ml of PBS. The cells were allowed to settle for 5 min, after which the top 2 ml were discarded. The filtrate was centrifuged at 1000 rpm for another 3 min, then the cell pellet was resuspended in D-MEM/F12 medium. The isolated endometrial cells were plated at a density of approximately 2–3 x10⁶ cell/ml onto floating, permeable supports made of nitrocellulose Millipore filters and a silicone ring (with a surface area of 0.45 cm² for cell growth). For pH_i measurement, cell suspension was plated onto floating, permeable supports made of Transwell-Col membranes (Costar, Cambridge, MA) with a cell growth area of 0.1 cm². Each filter/membrane was coated with 100/40 µl of Matrigel diluted 8 times using PBS. Cultures were floated on medium in Petri dishes and incubated at 37°C in 95% O₂/5% CO₂ for 3 days until the cells formed a confluent monolayer, which was polarized epithelium.

RNA Isolation and RT-PCR

Total RNA was isolated from immature mouse uterus 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. In this study, NBC primers for PCR reactions were based on the sequences of mouse NBC (AF020195) [30]. The primers were 5'-TGGAGCAAACCCCATGTGGCC-3' (sense) and 5'-CACCGCAGAACCGGCCAGTTC-3' (antisense), which generated a 901-base pair (bp) NBC PCR product. As for the negative control, RT-PCR was performed in the absence of reverse transcriptase. The conditions for PCR were as follows: 4 min at 94°C (initial melt); 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1.5 min at 72°C; and then 15 min at 72°C (final extension). The PCR products were analyzed by agarose gel electrophoresis with ethidium bromide.

I_SC Measurement

Measurement of the I_SC has been described previously [31, 23]. Briefly, monolayers grown on permeable supports were clamped vertically between 2 halves of an Ussing chamber (World Precision Instrument, Sarasota, FL). Electrodes for measuring transepithelial potential and Ag-AgCl current-passing electrodes were connected to the chamber by a salt bridge filled with 3 M KCl in 1.5% agar. Changes in the I_SC were recorded with a DVC-1000 voltage-current clamp amplifier (World Precision Instrument). Transepithelial resistance was obtained from the ohmic relationship by clamping the tissue intermittently at a value of 0.1 mV every 10 sec. The monolayers were bathed on both sides with Krebs-Henseleit (K-H) solution maintained at 37°C by a water jacket enclosing the reservoir. Pooled data were expressed as the increase in current per square centimeter of monolayer (µA/cm²). The K-H solution contained 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 24.8 mM NaHCO₃, 1.2 mM KH₂PO₄, and 11.1 mM glucose. To observe a net anion secretion, amiloride (10 µM), a Na⁺-channel blocker, was applied apically to exclude Na⁺ absorption [23]. In ion substitution experiments, ambient Cl^- or HCO₃^- was replaced by gluconate, and Na⁺ was replaced by NMDG⁺. The solution was bubbled with 95% O₂/5% CO₂ to maintain the pH of the solution at 7.4. When HCO₃^- was removed, the solution was gassed with 100% O₂. Substance could be added directly to the apical or the basolateral side of the epithelium.

pH_i Measurement

The samples were rinsed with NaCl solution and incubated with BCECF-AM (3 µM), a fluorescent dye, in standard NaCl solution for 30 min at 37°C. The sample was then mounted into a miniature Ussing chamber attached to the stage of the inverted microscope (model TE300; Nikon, Tokyo, Japan). Samples were superfused with prewarmed experimental solutions, and pH_i was measured with the PTI Ratio-Master fluorescence system (Photon Technology International, Lawrenceville, NJ). The intracellular dye was alternately excited at 2 wavelengths (440 and 490 nm), and the emission was measured at a wavelength of 510 nm by a photometer and then recorded. The calibration curve for pH_i was made according to the method of Thomas et al. [32]. In brief, cells were exposed to Hepes-buffered solution containing 120 mM K⁺ and 10 µM nigericin, and solution pH was adjusted at the different levels (from 6.4 to 7.8) with KOH. The standard NaCl solution contained 139.0 mM NaCl, 4.0 mM KCl, 10 mM Hepes, 17.5 mM D-glucose, 1.0 mM CaCl₂, 0.7 mM MgCl₂, and 1.0 mM NaHPO₄. For Na⁺-free solution, NaCl was replaced in equimolar concentrations by NMDG-Cl. For NH₄⁺ solution, 20 mM NH₄Cl replaced NaCl in equimolar concentrations. The Na⁺-HCO₃^- solution contained 119.0 mM NaCl, 4.0 mM KCl, 5 mM Hepes, 25.0 mM NaHCO₃, 17.5 mM D-glucose, 1.0 mM CaCl₂, 0.7 mM MgCl₂, and 1.0 mM NaHPO₄. The NaCl, Na⁺-HCO₃^-, and NH₄⁺ solutions were adjusted to pH 7.4 with NaOH. The NMDG-Cl solution was adjusted to pH 7.4 with KOH. The Na⁺-HCO₃^- solution was gassed with 95% O₂/5% CO₂ throughout the experiment.

Statistical Analysis

Current response was normally measured by the peak current magnitude. The change in I_SC was defined as the maximal rise in I_SC on stimulation. Data were normalized as current change per unit area of epithelial monolayer (µA/cm²). Results are expressed as the mean ± SEM, and the n value indicates the number of experiments. Comparisons between groups of data were made by Student paired or unpaired t-test. A P value of less than 0.05 was considered to be statistically significant.

	RESULTS

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Contribution of HCO₃^- to the Forskolin-Stimulated I_SC

Forskolin (10 µM), an adenylate cyclase activator, was used to stimulate anion secretion, because a number of physiological regulators, such as adrenaline and prostaglandins, had been shown previously to converge on a cAMP-dependent pathway in mouse endometrial epithelium [25]. When bathed in normal Cl^-- and HCO₃^--containing K-H solution in the presence of apical amiloride (10 µM) to eliminate Na⁺ absorption, the cultured endometrial epithelium responded to forskolin (10 µM) stimulation with an increase of 11.7 ± 0.5 µA/cm² (n = 18) in the I_SC (Fig. 1A). When Cl^- in the bathing solution was replaced by impermeant gluconate, the forskolin-stimulated I_SC was reduced by 55.6% (to 5.2 ± 0.6 µA/cm², n = 8) (Fig. 1B). Basolateral addition of bumetanide (100 µM), an inhibitor of Na⁺-K⁺-2Cl^- cotransporter, resulted in a 52.1% reduction in the forskolin-stimulated I_SC (to 5.6 ± 0.7 µA/cm², n = 8) (Fig. 1C). The result was similar to that obtained in Cl^--free solution, in which case Na⁺-K⁺-2Cl^- cotransporter would have been inactivated. The remaining forskolin-stimulated I_SC in both cases might be due to the contribution by HCO₃^-. Indeed, when both Cl^- and HCO₃^- were absent from bathing solution, the forskolin-induced I_SC was almost completely abolished, with a reduction of 96.6% in I_SC (to 0.4 ± 0.1 µA/cm², n = 5) (Fig. 1D). Similarly, in the presence of bumetanide and the absence of HCO₃^-, an 87.2% reduction in the I_SC (to 1.5 ± 0.4 µA/cm², n = 5) was observed (Fig. 1E). These results indicate that the forskolin-induced I_SC was contributed by both Cl^- and HCO₃^- secretion. A summary of these results is plotted in Figure 1F.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Effect of extracellular anion replacement and transport inhibition on forskolin-stimulated ISC. Representative recordings of the ISC activated by forskolin obtained in normal K-H solution (n = 18) are shown. A) Cl--free solution (n = 8). B) Bumetanide pretreated in normal K-H solution (n = 8). C) Cl-- and HCO3--free solution (n = 5). D) Bumetanide pretreated in HCO3--free solution (n = 8). E). Arrows mark the time at which forskolin (forsk; 10 µM) or bumetanide (bumet; 100 µM) was added basolaterally. Experiments were conducted with amiloride (10 µM) present in the apical solution. F) Summary of the results showing peak current magnitude under the different conditions stated above. Column and bars represent mean ± SEM.

Na⁺-Dependence of Forskolin-Stimulated Anion I_SC

Previous studies have demonstrated that replacing apical Na⁺ does not reduce the cAMP-activated I_SC, excluding the contribution of Na⁺ absorption to the cAMP-activated I_SC. However, the present study with bilateral Na⁺ replacement resulted in almost complete abolishment of the forskolin-stimulated I_SC (to 0.8 ± 0.4 µA/cm², n = 5) (Fig. 2). The presence of apical amiloride (10 µM), which should have excluded the possible involvement of Na⁺ absorption through apical Na⁺ channels, suggested that the effect of Na⁺ removal was due to basolateral Na⁺. This result, together with that obtained in Cl^-- and HCO₃^--free solution, suggest that both Cl^- and HCO₃^- secretion involve transport mechanisms that depend on basolateral Na⁺.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Na+-dependence of forskolin-stimulated ISC. A) Representative ISC recording of the forskolin-stimulated response in normal K-H solution and Na+-free solution. B) Summary of the results (n = 5). Column and bars = mean ± SEM.

Effect of HCO₃^- Substitution and NBC Inhibitor

Removing HCO₃^- alone from the bathing solution resulted in a 43.6% reduction in the forskolin-stimulated I_SC (from 11.7 ± 0.5 to 6.6 ± 0.4 µA/cm², n > 7, P < 0.001) (Fig. 3). The dependence of the forskolin-stimulated I_SC on both HCO₃^- and Na⁺ (see above) suggested possible involvement of NBC. To test this, H₂DIDS, an inhibitor of NBC, was used. Basolateral addition of H₂DIDS (450 µM) resulted in a 47.9% reduction in the forskolin-stimulated I_SC (to 6.1 ± 0.6 µA/cm², n = 6) (Fig. 3), similar to that induced by HCO₃^- removal. The inhibitory effect of H₂DIDS was also observed in Cl^--free solution (n = 4, P < 0.05) (Fig. 4) but was not significant in HCO₃^--free solution (n = 3, P > 0.05) (Fig. 5), confirming the effect of H₂DIDS on HCO₃^- transport, which most likely involved NBC.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Effect of HCO3- removal and H2DIDS on forskolin-stimulated ISC. Summary of the forskolin-stimulated ISC responses under different conditions (n 6) is shown. Column and bars = mean ± SEM

View larger version (12K): [in this window] [in a new window]   FIG. 4. Effect of H2DIDS on forskolin-induced ISC in Cl--free solution. A) Respective recordings of the forskolin-induced responses obtained in Cl--free solution without and with H2DIDS. B) Summary of results (n = 4). Column and bars = mean ± SEM

View larger version (13K): [in this window] [in a new window]   FIG. 5. Ineffectiveness of H2DIDS in HCO3--free solution. A) Respective recordings of forskolin-induced responses obtained in HCO3--free solution without and with H2DIDS in HCO3--free solution. B) Summary of the results (n = 3). Column and bars = mean ± SEM. Note that the effect of H2DIDS was not statistically significant.

pH_i Recovery from Cellular Acidification

To achieve transepithelial HCO₃^- secretion, accumulation of HCO₃^- into the cell from the basolateral compartment is necessary. The HCO₃^- accumulation should be accompanied by changes in pH_i. Therefore, measurement of pH_i recovery from NH₄Cl-induced cellular acidification was conducted to examine the possible involvement of NBC. After acidification, little pH_i recovery was observed when both Na⁺ and HCO₃^- were absent in the basolateral perfusion solution, but when Na⁺ and HCO₃^- were reintroduced into the perfusion solution, a rapid recovery of pH_i was observed (n = 5) (Fig. 6A). Because Na⁺/H⁺ exchanger (NHE), which is known to be involved in pH_i regulation, may contribute to pH_i recovery, amiloride (500 µM) was basolaterally added to eliminate the effect of NHE. As shown in Figure 6B, in the presence of amiloride, Na⁺ alone, without HCO₃^-, was not able to elicit the recovery of pH_i, and a rapid recovery of pH_i was observed only when HCO₃^--containing solution was also perfused (n = 6). However, basolateral HCO₃^- alone, without Na⁺, was not able to elicit recovery of pH_i (data not shown), suggesting that the HCO₃^- influx was mediated by NBC.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Basolateral Na+- and HCO3--dependence of pHi recovery from an acid load. Mouse endometrial cells were initially bathed in bilateral standard NaCl solution and then changed to basolateral NH4+ solution. NH4+ solution was subsequently replaced by bilateral Na+-free solution for cellular acid loading. A) Na+-free solution was replaced by Na+- and HCO3--containing solution in basolateral bath at the time indicated. The diagram is a representative tracing from 5 similar experiments. B) Na+-free solution was basolaterally replaced by standard NaCl solution with the presence of amiloride (Amil; 500 µM) and then transferred to Na+- and HCO3--containing solution with amiloride as indicated. The diagram is a representative tracing from 6 similar experiments. AP, Apical; BL, basolateral.

RT-PCR Demonstration of NBC Expression

The RT-PCR experiments were conducted to demonstrate the expression of NBC in mouse endometrial cells. A PCR product of a 901-bp fragment was observed (Fig. 7), as expected, using primers designed from the mouse sequence of NBC. Absence of the cDNA template did not give rise to any observable band, confirming that the PCR product was not due to nonspecific amplification. Sequencing of the PCR product confirmed the expression of NBC in mouse endometrial cells.

View larger version (57K): [in this window] [in a new window]   FIG. 7. RT-PCR analysis of NBC mRNA in mouse uterus. The PCR products are seen in reaction using oligonucleotide primer pairs for NBC (901 bp). Negative control was performed in the absence of relevant cDNA. The DNA markers are indicated on the left.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study investigated the basolateral mechanism underlying cAMP-activated HCO₃^- secretion across the mouse endometrial epithelium. The results of the present study have clearly demonstrated that the cultured mouse endometrial epithelial cells were capable of secreting HCO₃^-, with the basolaterally located NBC playing a key role in HCO₃^- accumulation.

The first line of evidence came from the I_SC: 1) when Cl^- secretory I_SC was excluded by ion substitution or by inhibition of Na⁺-K⁺-2Cl^- cotransporter, the remaining forskolin-stimulated I_SC was further abolished by removal of HCO₃^- from the bathing solution; 2) the forskolin-stimulated I_SC was inhibited by H₂DIDS, which could be observed even in the absence of extracellular Cl^- but not in the absence of HCO₃^-; and 3) the forskolin-induced I_SC was dependent on basolateral Na⁺. It should be noted that all I_SC experiments in the present study were conducted in the presence of apical amiloride. Therefore, the contribution of Na⁺ absorption, or of any effect caused by apical Na⁺, could be ignored. The results clearly showed that the forskolin-stimulated I_SC rise was mediated by both Cl^- and HCO₃^- secretion. At the same time, the forskolin-stimulated I_SC could be abolished by the replacement of bathing Na⁺, which suggests that both Cl^- and HCO₃^- secretion were dependent on basolateral Na⁺. This result suggested the presence of a basolateral transport mechanism transporting both Na⁺ and HCO₃^- ions, most likely the NBC. This was confirmed by the inhibition of the forskolin-induced HCO₃^--dependent I_SC by H₂DIDS, which has been shown to inhibit NBC in a number of tissues [14].

Further evidence supporting the involvement of NBC came from pH_i measurements. The pH_i recovery from cellular acidification could be mediated by either H⁺ extrusion or HCO₃^- influx. In the presence of basolateral amiloride at a concentration known to inhibit NHE, a major mechanism for pH_i regulation, the pH_i recovery was observed only when both Na⁺ and HCO₃^- were present in the basolateral compartment. This suggested that HCO₃^- influx through the basolateral membrane requires the presence of Na⁺. The results obtained from pH_i measurement are consistent with the involvement of NBC in cellular HCO₃^- accumulation through the basolateral membrane. The presence of NBC in mouse endometrial epithelium was further confirmed by the results of RT-PCR experiments.

A mechanism involving conversion of CO₂ into carbonic acid through the action of carbonic anhydrase has been suggested to contribute to the intracellular HCO₃^- accumulation in a number of tissues [33–35]. However, our studies have shown that it only plays a small role in mouse endometrium (unpublished data). Thus, basolaterally located NBC, as demonstrated in the present study, may play a key role in HCO₃^- accumulation in mouse endometrial epithelial cells.

The forskolin-stimulated (cAMP-activated) HCO₃^- secretion across the endometrial epithelium demonstrated in the present study suggests that the HCO₃^- content of the uterine fluid may be fine-tuned through neurohormonal regulation, because our previous studies have demonstrated that several neurohormonal regulatory pathways are cAMP-dependent [23–27]. Regulation of HCO₃^- secretion may modify the luminal pH of the uterus, which is required for the optimal function of many enzymes present in the uterine fluid. More importantly, HCO₃^- is known to play a crucial role in spermatozoa capacitation and embryo development [6, 7]. Regulated HCO₃^- secretion may provide the sufficiently high HCO₃^- content that is required for a particular reproductive event at a particular time.

It remains to be elucidated, however, whether NBC is directly subject to neurohormonal regulation or whether the regulation of HCO₃^- secretion is achieved through an elaborate operation of various cellular mechanisms. In the present study, we have demonstrated a mechanism, namely NBC, that is involved in HCO₃^- transport through the basolateral domain of the epithelium. It is not clear which cellular mechanism is responsible for apical secretion of HCO₃^-. Possible candidates may include anion exchanger (e.g., Cl^-/HCO₃^-) and anion channels [36, 37]. We have also obtained some evidence suggesting the involvement of CFTR, itself a cAMP-activated Cl^- channel. This notion is consistent with the cAMP-dependence of the HCO₃^- secretion as demonstrated in the present study. Together with the recently reported involvement of CFTR in regulation of a number of HCO₃^--related transport mechanisms [38], the presently demonstrated cAMP-activated HCO₃^- secretion may, considering the important role of HCO₃^- in various reproductive events occurring in the uterus, have bearing on the reduced fertility observed in women with cystic fibrosis.

In summary, the present study has demonstrated that basolaterally located NBC plays an important role in mediating HCO₃^- secretion across mouse endometrial epithelium. Further work on the details of the apical transport mechanism and regulation of HCO₃^- secretion may provide answers to some unexplained cases of infertility in women.

	FOOTNOTES

 
First decision: 19 October 2001.

¹ The work was carried out at the Epithelial Cell Biology Research Center and was supported by the direct grant (2040775) and strategic research program of The Chinese University of Hong Kong and National 973 Research Project of China.

² Correspondence. FAX: 852 2603 5022; hsiaocchan{at}cuhk.edu.hk

Accepted: January 11, 2002.

Received: September 17, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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