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Cellular Mechanisms of Adrenaline-Stimulated Anion Secretion by the Mouse Endometrial Epithelium¹

^a Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong ^b Shanghai Institute of Planned Parenthood Research, Shanghai, China

	ABSTRACT

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The uterine fluid composition is largely determined by the absorptive and secretory activities of the endometrial epithelium. The present study explored the cellular mechanisms involved in adrenaline-stimulated anion secretion across the cultured mouse endometrial epithelium using the short-circuit current (I_SC) technique in conjunction with transporter inhibitors and channel blockers. Cultured endometrial epithelial monolayers responded to basolateral application of adrenaline with an increase in I_SC, which was attributable to both Cl^- and HCO₃^- secretion. When extracellular Cl^- or HCO₃^- was removed, the adrenaline-induced response, as measured by the total charge transfer per unit area, was reduced to 53% and 46%, respectively. When both Cl^- and HCO₃^- were absent from the bathing solutions, the adrenaline-induced response was reduced to only 2% of the response when both ions were present, indicating substantial contribution of Cl^- and HCO₃^- secretion to the adrenaline-stimulated response. Cellular mechanisms, e.g., transporters and ion channels, involved in Cl^- or HCO₃^- secretion were investigated separately. Cl^- secretion was found to depend on the activities of basolaterally located Na⁺-K⁺-ATPase, Na⁺-K⁺-2Cl^- cotransporter, and K⁺ channels, while evidence suggested that HCO₃^- secretion depends substantially on basolaterally situated Na⁺-HCO₃^- cotransporter and Na⁺-H⁺ exchanger. Similar to what was seen for Cl^- exit, a large portion of HCO₃^- appeared to exit apically through anion channels. The results indicate that the uterine fluid composition in the mouse may be regulated by adrenaline through stimulation of both Cl^- and HCO₃^- secretion and may be fine-tuned through an elaborate operation of different cellular mechanisms.

	INTRODUCTION

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Measurements of human uterine fluid composition have revealed active ion transport across the endometrium through absorptive and secretory activities [1]. Changes in the ionic composition of uterine fluid, especially Cl^- and HCO₃^-, during various reproductive events including implantation have been observed in a number of species [2–4]. The physiological importance of the ionic composition of uterine fluid may by emphasized by its influence on sperm motility, embryo viability, and development.

The endometrial epithelium is thought to be largely responsible for the formation of uterine fluid, since epithelial cells function in transcellular transport of fluid, ions, and diffusible molecules and thereby modulate the tonicity and pH of luminal fluids. In contrast to the situation for other epithelial tissues, however, the transport properties of endometrial epithelial cells have not been adequately studied. Although it has been reported that various neurohormonal agents including adrenaline, bradykinin, bombesin, gastrin-releasing peptide, and prostaglandin E₂ and F₂ are effective stimulants of electrogenic transport across endometrial epithelia of a number of species [5–13], the cellular mechanisms, e.g., transporters and ion channels, underlying these responses have not been elucidated.

Recently, a primary culture of mouse endometrial epithelial cells grown on permeable supports has been established and shown to have a basal short-circuit current (I_SC) predominantly mediated by Na⁺ absorption [14]. It has also been demonstrated that the cultured epithelium responds to adrenaline or noradrenaline with increases in the I_SC that can be predominantly attributable to Cl^- and HCO₃^- secretion [15]. These secretory responses seem to be mediated by intracellular cAMP through ß-adrenoceptor activation. The adrenaline-induced I_SC could be inhibited by pretreatment with diphenylamine 2,2'-dicarboxylic acid or replacement of external Cl^- and HCO₃^-, but not by amiloride or replacement of Na⁺ in apical solution.

In the present study, we further clarified the cellular mechanisms of adrenaline-stimulated anion secretion by cultured mouse endometrial epithelium using ion substitution, selective transporter inhibitors, and ion channel blockers in conjunction with the I_SC technique. The results suggest that various cellular mechanisms are involved in the adrenaline-stimulated Cl^- and HCO₃^- secretion by the mouse endometrial epithelium.

	MATERIALS AND METHODS

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Materials

Adrenaline was obtained from David Bull Laboratories (Lexia Place Mulgrave, Victoria, Australia). Dulbecco's Modified Eagle's medium (DMEM), F-12 nutrient mixture, penicillin-streptomycin, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), acetazolamide, bumetanide, ouabain, and methyl isobutyl amiloride (MIA) were purchased from Sigma Chemical Co. (St. Louis, MO). Dihydrogen-4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (H₂DIDS) was purchased from Molecular Probes (Eugene, OR). Diphenylamine-2,2'-dicarboxylic acid (DPC) was obtained from Riedel-de Haen Chemicals (Hannover, Germany).

Cell Isolation and Culture

Endometrial epithelial cells were enzymatically isolated from the mouse uterus according to the method described by McCormack and Glasser [16] with slight modification [14]. For each culture preparation, samples of uteri were obtained from 40 immature ICR mice (3.5–4 wk) to avoid complications of the estrous cycle. The sliced uteri were incubated in PBS supplemented with 5 mg/ml trypsin, 25 mg/ml pancreatin, 100 U/ml penicillin, and 100 mg/ml streptomycin at 0°C for 60 min and then at room temperature for another 60 min. After enzyme digestion, the test tube containing PBS and the tissues was shaken gently for 30 sec. Uterine tissues were carefully removed, and the crude cell solution was passed through a 70-µm fluorocarbon filter from Spectrum (Houston, TX). The filtrate was centrifuged at 1000 x g for 5 min. The cell pellet was resuspended in 12 ml PBS and centrifuged again at 1000 x g for 5 min. The washing procedures were then repeated once more. After centrifugation, the cell pellet was resuspended in DMEM/Ham's F-12 culture medium containing 10% fetal bovine serum, 1% non-essential amino acid, and 100 U/ml penicillin plus 100 mg/ml streptomycin. The isolated endometrial cells were plated at a density of about 1.4 x 10⁶ cells/ml onto floating permeable supports made of nitrocellulose Millipore (Bedford, MA) filter and a silicone ring (with a surface area of 0.45 cm² for cell growth). Cultures were incubated at 37°C with 5% CO₂:95% O₂ gas atmosphere and reached confluence in 3 days. The cells grown on permeable supports formed polarized epithelia [14].

I_SC Measurement

The measurement of I_SC has been described previously [17, 18]. Monolayers grown on permeable supports were clamped vertically between two halves of the Ussing chamber. Monolayers were bathed on both sides with Krebs-Henseleit (K-H) solution, which was maintained at 37°C by a water jacket enclosing the reservoir. K-H solution had the following composition: 117 mM NaCl; 4.5 mM KCl; 2.5 mM CaCl₂; 1.2 mM MgSO₄; 24.8 mM NaHCO₃; 1.2 mM KH₂PO₄; 11.1 mM glucose. In some experiments, ambient Cl^- and/or HCO₃^- was removed by substituting with gluconate. The solution was bubbled with 95% O₂:5% CO₂ such that the pH of the solution was maintained at 7.4. When HCO₃^- was removed, the solution was gassed with 100% O₂. Substances could be added directly to the apical or basolateral side of the epithelium. The epithelium exhibited a basal transepithelial potential difference (PD) for every monolayer examined, which was connected to a voltage-clamp amplifier (DVC-1000; World Precision Instruments, Sarasota, FL). In each experiment, a transepithelial PD of 0.1 mV was applied. The change in current in response to the applied potential was used to calculate the transepithelial resistance of the monolayer using the Ohmic relationship.

Data 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 upon agonist stimulation. Data were normalized as current change per unit area of epithelial monolayer (µA/cm²). However, in experiments with pretreatment of inhibitors, some of the transport inhibitors drastically reduced the duration of the adrenaline-induced response but not the peak magnitude of the I_SC response. As a result, it is much informative and appropriate to compare the area under the I_SC response curve, the total charge transfer across the epithelium, than to compare the peak amplitude of the I_SC response. Arbitrarily, the area under the curve over a 30-min period (A) was measured, and the total charge transfer can be calculated according the following simple equation: Total charge transfer (µC) = A x 150.

One square centimeter of the measured area under the I_SC response curve corresponds to 0.5 µA x 300 sec or 150 µC. The area of the permeable membrane confined by silicone, where the endometrial epithelial cells were held, was 0.45 cm² in size; thus the total charge transfer per unit area of epithelium would be determined by the following equation: Total charge transfer per unit area (µC/cm²) = A x 150 ÷ 0.45.

For those experiments in which inhibitors were added to the cell monolayers after the stimulation with adrenaline, the reduction in I_SC response was measured and percentage inhibition calculated. The dose of adrenaline used was 1 µM throughout the present study, as this is the minimum dose causing the maximum I_SC response as deduced from the concentration-response curves [15].

Results are expressed as mean ± SE. Comparisons between groups of data were made by Student's unpaired t-test. A reference p value of less than 0.05 was considered statistically significant.

	RESULTS

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I_SC Responses to Adrenaline

The polarized layers of mouse endometrial epithelium used in the present study had a transepithelial electrical resistance of 730 ± 84 cm² (n = 49), with a basal I_SC (I_b) of 4.3 ± 0.2 µA/cm² (n = 49) in the unstimulated state when bathed in Cl^-/HCO₃^--containing solution. The cultured mouse endometrial epithelium responded to adrenaline (1 µM, basolateral) differently, in both magnitude and duration of the response, when bathed in solutions containing different anions (Fig. 1). Note that the shape of the adrenaline-stimulated I_SC response obtained from samples bathed in HCO₃^--free solution (panel C) is different from that of monolayers bathed in Cl^-/HCO₃^--containing solution (panel A) and Cl^--free solution (panel B); two consecutive peaks could be observed in the HCO₃-free solution. The frequency of observation of this was about 70% (n = 21) among monolayers bathed in HCO₃^--free solution. A response of this shape was sometimes observed among samples bathed in Cl^-/HCO₃^--containing solution but never among those bathed in Cl^--free solution.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Effect of extracellular anion replacement on adrenaline-stimulated ISC. Representative recordings of the ISC activated by adrenaline (Adr, 1 µM) in normal K-H solution (A), Cl--free solution (B), HCO3--free solution (C), and Cl-- and HCO3--free solution (D), with arrows marking the extent of the basal current (Ib) and the time at which adrenaline was added basolaterally.

Relative Contribution of Cl^- and HCO₃^- to the Adrenaline-Stimulated I_SC

The relative contribution of Cl^- and HCO₃^- to the adrenaline-induced I_SC was assessed by the mean total charge transfer per unit area across the epithelial cell monolayers. Under stimulation with adrenaline, values of 5710 ± 230 µC/cm² (n = 15), 3030 ± 220 µC/cm² (n = 10), 2650 ± 270 µC/cm² (n = 15), and 130 ± 20 µC/cm² (n = 10) were for monolayers bathed in Cl^-- and HCO₃^--containing, Cl^--free, HCO₃^--free, and Cl^-- and HCO₃^--free solutions, respectively. The results are schematically represented in Figure 2. The total charge transfer per unit area, as well as the mean peak magnitude of I_SC responses in different bathing solutions, is plotted for comparison. Compared to the data obtained from cells bathed in normal K-H solution, replacement of Cl^- significantly reduced the total charge transfer per unit area to 53% (p < 0.001) and the peak magnitude of I_SC response to 68% (p < 0.001); replacement of HCO₃^- significantly reduced the total charge transfer per unit area to 46% (p < 0.001) and the mean peak magnitude of I_SC response to 60% (p < 0.001), while only 2% of the total charge transfer per unit area and 15% of the peak response remained when both Cl^- and HCO₃^- were replaced. Although there were slight differences in the results obtained using the two measurements, both the peak magnitude and the total charge transfer indicated that Cl^- and HCO₃^- contributed substantially to the adrenaline-induced I_SC.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Comparison of the total charge transfer per unit area (A) and the peak magnitude of ISC (B) induced by adrenaline in different bathing solutions. Note that the percentage reduction of total charge transfer by anion replacement in each experiment is greater than that of peak magnitude of ISC. Columns and bars are mean ± SE; data for each column were obtained from at least 10 independent experiments; p < 0.001 in all columns compared to the control.

Cellular Mechanism for Cl^- Secretion

As reported previously, bilateral replacement of Na⁺ almost completely abolished the adrenaline-induced I_SC, while apical replacement of external Na⁺ did not reduce the adrenaline-stimulated I_SC [15]. It seemed possible that a basolateral Na⁺-coupled mechanism is involved in the secretory process in the mouse endometrium. The most possible candidate was the Na⁺-K⁺-2Cl^- cotransporter. To verify this possibility, bumetanide (100 µM), an inhibitor of the Na⁺-K⁺-2Cl^- cotransporter, was applied basolaterally to the epithelial cell monolayers 10 min before the addition of adrenaline (1 µM). The pretreatment significantly reduced the adrenaline-induced I_SC in the normal bathing solution (Cl^-/HCO₃^--containing saline solution) as indicated by a 35% reduction of the total charge transfer (3810 ± 340 µC, n = 3) with bumetanide compared to the control value of 5900 ± 250 µC (n = 3; p < 0.001, Fig. 3). However, bumetanide was found to exert insignificant effect when external Cl^- was removed (n = 3, not shown), indicating that the effect of bumetanide was on a Cl^--dependent process. These results supported the involvement of a basolaterally located Na⁺-K⁺-2Cl^- cotransporter in mediating the Cl^--dependent adrenaline-induced I_SC.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Effect of bumetanide on the adrenaline-induced ISC in Cl-/HCO3--containing solution. A) Representative ISC recording (n = 3) of the adrenaline-stimulated response after treatment with bumetanide, with arrows marking the times at which basolateral bumetanide (Bum, 100 µM) or adrenaline (Adr, 1 µM) was added. B) Summary of the effect of bumetanide. Columns and bars are means ± SE.

The operation of Na⁺-K⁺-2Cl^- cotransporter requires a transmembrane Na⁺ gradient that is likely to be established by sodium potassium-adenosine triphosphatase (Na⁺-K⁺-ATPase). Thus, the effect of ouabain, an inhibitor of Na⁺-K⁺-ATPase, on the adrenaline-induced I_SC was also examined. Basolateral addition of ouabain (1 mM) to the adrenaline-stimulated monolayers resulted in a complete reversal of the adrenaline-induced I_SC responses (n = 4, Fig. 4A), indicating the presence of Na⁺-K⁺-ATPase in the basolateral domain.

View larger version (10K): [in this window] [in a new window]   FIG. 4. Effect of ouabain and barium on the adrenaline-induced ISC in Cl-/HCO3--containing solution. A) Representative ISC recording (n = 4) with arrows marking the time at which basolateral adrenaline (Adr, 1 µM) or ouabain (1 mM) was added. B) ISC recording (n = 6) with arrows marking the times at which basolateral adrenaline (Adr, 1 µM) or barium (Ba, 1 mM) was added.

The involvement of basolaterally located K⁺ channels was also investigated, since they play an important role in secondary active Cl^- transport. Basolateral addition of Ba²⁺ (1 mM), a K⁺ channel blocker, after adrenaline stimulation totally reversed the adrenaline stimulation of I_SC (n = 6, Fig. 4B). The inhibitory effect of Ba²⁺ was not observed in Cl^--free solution (n = 4, not shown), indicating that blocking of K⁺ channels by Ba²⁺ affects Cl^- secretion.

Cellular Mechanisms for HCO₃^- Secretion

In other HCO₃^- secretory systems like pancreatic duct cells, it is generally accepted that HCO₃^- secretion requires the conversion of CO₂ into carbonic acid through the action of carbonic anhydrase (CA). Does CA also play a role in the HCO₃^- secretion in the endometrial epithelial cells? To investigate this question, acetazolamide, a CA inhibitor, was used. After stimulation with adrenaline (1 µM), acetazolamide (45 µM) was added basolaterally to the monolayers bathed in Cl^-/HCO₃^--containing or Cl^--free solution and was found to induce similar reductions in the adrenaline-stimulated I_SC, 10.1 ± 2.0% (n = 6) and 12.8 ± 3.7% (n = 3), respectively (Fig. 5). In contrast, acetazolamide did not exert any effect on the adrenaline-stimulated I_SC when the monolayers were bathed in HCO₃^--free solution (n = 6, not shown). These results indicated a role of CA in the endometrial epithelial HCO₃^- secretion. Its contribution, however, is relatively small.

View larger version (11K): [in this window] [in a new window]   FIG. 5. Effect of acetazolamide on the adrenaline-stimulated ISC in Cl-/HCO3--containing solution. Representative ISC recording (n = 6) with arrows marking the times at which basolateral adrenaline (Adr, 1 µM), acetazolamide (Acet, 45 µM), and DPC (2 mM) were added.

Because a large portion of the HCO₃^- secretion is not dependent on CA, it remained possible that HCO₃^- ions enter endometrial epithelial cells through another HCO₃^- cotransporter situated at the basolateral domain; otherwise, HCO₃^- secretion would not be possible. The most likely candidate was the Na⁺-HCO₃^- cotransporter, which has been shown to be involved in HCO₃^- accumulation in other epithelial tissues [19–21]. To verify this, an inhibitor of Na⁺-HCO₃^- cotransporter, H₂DIDS, was used. Bathed in Cl^-/HCO₃^--containing solutions, cell monolayers pretreated with 0.45 mM H₂DIDS at the basolateral side showed an 80% reduction of the total charge transfer per unit area under adrenaline stimulation (1180 ± 60 µC, n = 4) as compared with the control value of 5700 ± 260 µC (n = 4; p < 0.001, Fig. 6). This result supported the notion that Na⁺-HCO₃^- cotransporter is involved in HCO₃^- uptake from the basolateral domain and thus plays an important role in the HCO₃^- secretion.

View larger version (17K): [in this window] [in a new window]   FIG. 6. Effect of H2DIDS on the adrenaline-stimulated ISC in Cl-/HCO3--containing solution. A) Representative ISC recording (n = 4) of the adrenaline-stimulated response after treatment with H2DIDS, with arrows marking the times at which basolateral H2DIDS (450 µM) or adrenaline (Adr, 1 µM) was added. B) Summary of the effect of H2DIDS. Columns and bars are means ± SE.

In order to sustain HCO₃^- secretion, H⁺ must be expelled from the cell. This may be achieved by the action of Na⁺-H⁺ exchanger. Basolateral pretreatment of the cell monolayers with MIA (100 µM), a specific Na⁺-H⁺ exchanger inhibitor, resulted in a 94% inhibition of the adrenaline-stimulated I_SC response in Cl^-/HCO₃^--containing solution (Fig. 7); the total charge transfer per unit area was 350 ± 20 µC (n = 3), compared to the control value of 5700 ± 260 µC (n = 4, p < 0.001), indicating the involvement of Na⁺-H⁺ exchanger in HCO₃^- secretion.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Effect of MIA on the adrenaline-stimulated ISC in Cl-/HCO3--containing solution. A) Representative ISC recording (n = 3) of the adrenaline-stimulated response after treatment with MIA, with arrows marking the times at which basolateral MIA (100 µM) or adrenaline (Adr, 1 µM) was added. B) Summary of the effect of MIA. Columns and bars are means ± SE.

The next question was how HCO₃^- ions exit the cell from the apical domain. Earlier experiments [15] had shown that adrenaline-stimulated I_SC could be blocked by apical application of Cl^- channel blockers. In the present study, experiments using Cl^- channel blockers were also conducted in Cl^--free solution to investigate the cellular mechanism for HCO₃^- exit. Apical application of DPC (1 mM) resulted in a significant reversal of the HCO₃^--dependent adrenaline-induced I_SC (84.7± 1.6%, n = 3; p < 0.001), as shown in Figure 8A. Another Cl^- channel blocker, DIDS, had a smaller effect on the HCO₃^--dependent adrenaline-stimulated I_SC, producing a maximal reversal of 17.2 ± 0.9% at 100 µM (n = 3; p < 0.05, Fig. 8B). These results suggest a major role of anion channels in mediating HCO₃^- exit across the mouse endometrial epithelium.

View larger version (11K): [in this window] [in a new window]   FIG. 8. Effect of Cl- channel blockers on the adrenaline-stimulated ISC in Cl--free solution. A) ISC recording (n = 3) with arrows marking the times at which basolateral adrenaline (Adr, 1 µM) or DPC (1 mM) was added. B) ISC recording (n = 3) with arrows marking the times at which basolateral adrenaline (Adr, 1 µM) or DIDS (100 µM) was added.

	DISCUSSION

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The present study is the first attempt to elucidate the cellular mechanisms underlying hormone-regulated anion secretion across the endometrial epithelium. The present study has demonstrated a secondary active transport mechanism for Cl^- secretion in endometrial epithelial cells. Several lines of evidence support this mechanism. First, pretreatment of the cells with the Na⁺-K⁺-2Cl^- cotransporter inhibitor, bumetanide, significantly inhibited the I_SC; however, the bumetanide effect was not observed when external Cl^- was replaced. These findings suggest that the action of bumetanide is on a Cl^--dependent process that most likely involves the Na⁺-K⁺-2Cl^- cotransport system. Secondly, basolateral addition of the Na⁺-K⁺-ATPase inhibitor, ouabain, totally inhibited the adrenaline-stimulated I_SC, indicating the presence of Na⁺-K⁺-ATPase. This is required to sustain a Na⁺ gradient necessary for driving the Na⁺-K⁺-2Cl^- cotransporter. Lastly, the application of Ba²⁺, a K⁺ channel blocker, to the basolateral side of the cells completely inhibited the adrenaline-induced I_SC; however, it was without effect when external Cl^- was replaced, indicating that the Cl^- secretion was largely dependent on basolateral K⁺ channels. Basolateral K⁺ channels provide a pathway for the recycling of K⁺, which is crucial for the maintenance of both Na⁺-K⁺-ATPase and Na⁺-K⁺-2Cl^- cotransporter. Together these basolaterally localized transporters are responsible for the accumulation of Cl^- above its electrochemical equilibrium inside the epithelial cells. Cl^- can then exit the cells through the apical glibenclamide- and DPC-sensitive Cl^- channels as reported previously [15]. The involvement of these transporters in mediating adrenaline-stimulated Cl^- secretion across the mouse endometrial epithelium indicates a secondary active transport mechanism that has been well documented to be responsible for Cl^- secretion in many other epithelia [18, 22–24]. In accord with the evidence mentioned above, Na⁺-K⁺-ATPase has been visualized in the surface epithelium and glands in the mouse uterus by immunocytochemistry [25]. Those investigators reported a uniform distribution and moderate abundance of Na⁺-K⁺-ATPase that was confined to the basal and lateral plasma membrane. Confirmation of the presence of other transporters awaits future localization studies.

The present study has also demonstrated that the adrenaline-stimulated HCO₃^- secretion depends largely on basolaterally situated Na⁺-HCO₃^- cotransporter and Na⁺-H⁺ exchanger. This notion is supported by the following observations: 1) CA inhibitor, acetazolamide, blocked only about 10% of the adrenaline-induced I_SC in Cl^-/HCO₃^--containing or Cl^--free solution, indicating that intracellular conversion of bicarbonate from CO₂ and H₂O makes a limited contribution to adrenaline-induced HCO₃^- secretion, and thus, that a mechanism for HCO₃^- uptake at the basolateral domain would be necessary; 2) basolateral pretreatment of the epithelial cells with an inhibitor of Na⁺-HCO₃^- cotransporter, H₂DIDS, resulted in an 80% reduction of the adrenaline-stimulated I_SC response; and 3) basolateral pretreatment of the monolayers with MIA, a specific Na⁺-H⁺ exchanger inhibitor, resulted in over 90% inhibition of the I_SC response to adrenaline. While Na⁺-HCO₃^- cotransporter is responsible for the uptake of HCO₃^- into the cells, Na⁺-H⁺ exchanger expels H⁺ from the cells to achieve maximum HCO₃^- accumulation.

The presence of a Na⁺-dependent HCO₃^- transport mechanism, in addition to the Na⁺-dependent Cl^- secretion, could explain why more than 95% inhibition of adrenaline-induced I_SC was observed when external Na⁺ was removed [14] but only 35% inhibition was observed with bumetanide treatment. Bumetanide would only abolish Na⁺-dependent Cl^- secretion, leaving HCO₃^- secretion intact, while removal of external Na⁺ suppresses both types of secretion if both are Na⁺-dependent. In other words, bumetanide-sensitive Na⁺-K⁺-2Cl^- cotransporter is not the only Na⁺-dependent mechanism involved in the adrenaline-induced I_SC response. HCO₃^- secretion triggered by adrenaline is also Na⁺-dependent. This is most likely mediated by the Na⁺-HCO₃^- cotransporter. One could argue that the observed inhibitory effect of H₂DIDS was on Cl^--HCO₃^- exchanger, rather than Na⁺-HCO₃^- cotransporter, since H₂DIDS may also block the activity of the Cl^--HCO₃^- exchanger. This possibility is excluded by the morphological observation that Cl^--HCO₃^- exchanger is absent from the endometrial epithelium as well as other tissues of the genital tract of female rodents [25].

The overwhelmingly large percentage of inhibition of the adrenaline response by the inhibitors of Na⁺-HCO₃^- cotransporter and Na⁺-H⁺ exchanger, H₂DIDS and MIA, respectively, may seem a bit puzzling. It should be noted that intracellular pH would be disturbed by the two inhibitors, since Na⁺-H⁺ exchanger has been proposed to be primarily responsible for the regulation of intracellular pH [26]. Similarly, inhibition of the Na⁺-HCO₃^- cotransporter may also exert an effect on intracellular pH. Therefore, Cl^- secretion could be indirectly affected by upsetting of intracellular pH regulation. It should be noted that some of the inhibitors used may have nonspecific effects other than their expected action on transporters. Therefore, further investigations using immunohistochemical techniques are required to confirm the existence of individual transporters.

The presently demonstrated adrenaline-regulated Cl^- and HCO₃^- secretion across the endometrial epithelium suggests that uterine fluid composition may be delicately fine-tuned through neurohormonal regulation and the elaborate operation of various cellular mechanisms. While uterine fluid driven by anion secretion, as seen in many epithelia, may be required for sperm function and embryo development, HCO₃^- secretion may modify the pH of the uterine lumen. Since many enzymes known to be involved in implantation [3, 4] require optimal pH for their action, the importance of regulated anion secretion, particularly HCO₃^- secretion, by the endometrial epithelium is thus easy to envisage. It should be mentioned that prostaglandin E₂, which is known to play a crucial role in implantation, has also been shown to stimulate both HCO₃^- and Cl^- secretion across the endometrial epithelium [27], further indicating the importance of the secretion of these anions in uterine functions. Our data suggest that hormone-regulated anion secretion may constitute the physiological basis for the observed high pH and HCO₃^- content in the rabbit uterus [28] and the increased Cl^- concentration during implantation in the rat [29].

In summary, the present results indicate that uterine fluid composition in the mouse may be regulated by adrenaline through stimulation of both Cl^- and HCO₃^- secretion and fine-tuned through an elaborate operation of different cellular mechanisms. A similar effect of adrenaline has also been reported for the oviduct [30]. These observations taken together, adrenaline-regulated anion secretion may be important for the normal function of the female reproductive tract.

	FOOTNOTES

 
¹ Supported by the Research Grants Council of Hong Kong.

² Correspondence. FAX: 825 2603 5022; hsiaocchan{at}cuhk.edu.hk

Accepted: July 21, 1998.

Received: February 17, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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