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Adenosine Triphosphate Induces Inhibition of Na⁺ Absorption in Mouse Endometrial Epithelium: A Ca²⁺-Dependent Mechanism¹

^a Epithelial Cell Biology Research Centre, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong

ABSTRACT

The present study investigated the inhibitory effect of extracellular ATP on Na⁺ absorption and the possible underlying mechanism in cultured mouse endometrial epithelium using the short-circuit current (I_SC) technique. The cultured epithelia exhibited a Na⁺-dependent basal current that could be predominately blocked by the epithelial Na⁺ channel (ENaC) blocker, amiloride (10 µM). Apical addition of ATP (10 µM) induced a reduction in basal I_SC. However, in the presence of amiloride or when apical Na⁺ was removed, the ATP-induced reduction was abolished and an increase in the I_SC was observed with kinetic characteristics similar to those reported previously for the ATP-induced Cl^- secretion, indicating that ATP could induce both Cl^- secretion and inhibition of Na⁺ absorption. Further reduction in I_SC after ATP challenge could be obtained with forskolin (10 µM), which indicates that different inhibitory mechanisms are involved. The ATP-induced inhibition of Na⁺ absorption, but not that induced by forskolin, could be abolished by the P₂ receptor antagonist, reactive blue (100 µM), indicating the involvement of a P₂ receptor in mediating the ATP response. ATP and uridine 5'-diphosphate (UDP; 100 µM), a relatively selective agonist for the pyrimidinoceptor, induced separate I_SC reduction, and distinct I_SC increases in the presence of amiloride, regardless of the order of drug administration, indicating the involvement of two receptor populations. The ATP-induced inhibition of Na⁺ absorption was mimicked by the Ca²⁺ ionophore, ionomycin (1 µM), whereas the Ca²⁺ chelators, EGTA and BAPTA-AM, abolished the ATP-induced, but not the forskolin-induced, inhibition of Na⁺ absorption, suggesting the involvement of a Ca²⁺-dependent pathway. In the presence of the Cl^- channel blocker, DIDS (100 µM), both inhibitory and stimulatory responses to ATP were abolished, suggesting the involvement of a Ca²⁺-activated Cl^- channels (CaCCs) in mediating both ATP responses. The ATP-induced as well as the forskolin-induced reduction in I_SC was not observed when Cl^- was removed from the bathing solution, indicating that Cl^- permeation is important for the inhibition of Na⁺ absorption. The results suggest the presence of a Ca²⁺-dependent ENaC-inhibiting mechanism involving CaCC in mouse endometrial epithelial cells. Thus, extracellular nucleotides may play an important role in the fine-tuning of the uterine fluid microenvironment by regulating both Cl^- secretion and Na⁺ absorption across the endometrium.

calcium, signal transducers, signal transduction, uterus

INTRODUCTION

The uterine fluid microenvironment is believed to be important for sperm capacitation, embryo development, and implantation. However, the mechanisms underlying the regulation of uterine fluid remain largely unknown. Although changes in uterine fluid volume as well as its ionic composition have been reported in humans [1] and other species throughout the estrous cycle [2, 3], it is not clear how these changes are actually brought about. We have previously demonstrated active ion transport across cultured mouse endometrial epithelia, with the absorption of Na⁺ predominating in basal conditions, which can be switched to predominately anion secretion upon stimulation [4–7]. Like many epithelia, the net result of these two opposing processes determines the uterine luminal fluid ionic composition and volume. However, the mechanism regulating these processes and governing the switching is far from understood, especially in the uterus.

It has been shown that the activation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl^- channel, inhibits epithelial Na⁺ channels (ENaC) [8]. This has been considered as one of the mechanisms underlying the switching from Na⁺ absorption to Cl^- secretion. Recently, extracellular ATP, in addition to stimulating Cl^- secretion, has also been shown to induce inhibition of Na⁺ absorption in a number of epithelia [9–12], however, the signaling pathways involved in mediating the inhibitory effect of ATP have not been fully elucidated.

Recent evidence has indicated a paracrine and autocrine role of extracellular ATP and other nucleotides such as uridine 5' triphosphate (UTP) and uridine 5' diphosphate (UDP) in a variety of epithelia. The large amount of ATP contained in the sperm and semen also represents a physiological source of extracellular nucleotides in the uterine tract. The action of extracellular nucleotides could be mediated by a spectrum of receptors that can be classified into two major receptor families, the ligand-gated P2X receptors, and G protein-coupled P2Y receptors. Our previous study [4] on cultured mouse endometrial epithelium has demonstrated the activation of Cl^- secretion by extracellular ATP via the P_2Y2 (P_2U) receptor and Ca²⁺-dependent pathway involving the Ca²⁺-activated Cl^- channel (CaCC). Recent study has also revealed the involvement of a UDP-selective pyrimidinoceptor in ATP-induced endometrial Cl^- secretion. In order to investigate the inhibitory effect of ATP on Na⁺ absorption, the culture conditions have been manipulated to maximize the expression of ENaC (unpublished data) so that inhibition of Na⁺ absorption can be readily observed. In the present study, we compared the signaling pathways involved in ATP-induced Cl^- secretion and inhibition of Na⁺ absorption in cultured mouse endometrial epithelium using the short-circuit current (I_SC) technique.

MATERIALS AND METHODS

Materials

Dulbeccos modified Eagles medium (DMEM) with nutrient mixture F-12 (DMEM/F-12), PBS, fetal bovine serum (FBS), nonessential amino acids, and pancreatin were purchased from Gibco (Grand Island, NY). Penicillin/streptomycin and trypsin (type II), adenosine 5'-triphosphate (ATP), 1,2-bis-(2-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) acetoxymethyl ester (AM; BAPTA-AM), forskolin, UDP, ionomycin, EGTA, and 4,-4'-didsothiocyanostilbene-2,2'-disulfonic acid (DIDS) were obtained from Sigma Chemical Company (St. Louis, MO). Amilorode hydrochloride (reactive blue) was obtained from Research Biochemicals International (Natick, MA). Diphenylamine-2,2'-dicarboxylic acid (DPC) was purchased from Riedel de Haen Chemicals (Hannover, Germany). 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 McCormack and Glasser [13] with slight modifications [5]. Uteri were obtained from 3.5- to 4-wk-old immature ICR mice to avoid the complication of the estrous cycle. Isolated uteri 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 trypsin and 25 mg/ml pancreatin at 0°C for 60 min at room temperature for another 45 min. As the enzyme containing PBS was carefully poured off, DMEM/F-12 culture medium containing 10% FBS, 1% 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 after 5 min. The tissue was then gently shaken for 30 sec. Uterine tissue was removed and the filtrate was centrifuged at 1000 x g for 3 min. The supernatant was discarded and the cell pellet was resuspended in 12 ml PBS. The cells were allowed to settle for 5 min and the top 2 ml was discarded. The filtrate was centrifuged at 1000 x g for another 3 min, then the cell pellet was resuspended in DMEM/F-12 medium. The isolated endometrial cells were plated at a density of 2 to 3 x 10⁶ cell/ml onto the Matrigel-coated nitrocellulose Millipore filters (0.45 cm²). Each Millipore filter was coated with 100 µl of Matrigel diluted 8 times using PBS. Cultures were incubated at 37°C in 95% O₂/5% CO₂ and reached confluence in 3 days.

Short Circuit Current Measurement

The measurement of I_SC has been described previously [4, 14]. Monolayers grown on permeable supports were vertically clamped between 2 halves of the Ussing chamber. The monolayers were bathed on Krebs-Henseleit (K-H) solution that was maintained at 37°C by a water jacket that enclosed the reservoir. The K-H solution had the following composition: NaCl, 117 mM; KCl, 4.7 mM; CaCl₂, 2.5 mM; MgSO₄, 1.2 mM; NaHCO₃, 24.8 mM; KH₂PO₄, 1.2 mM; and glucose, 11.1 mM. In some experiments, ambient Cl^- was replaced by gluconate, and Na⁺ by NMDG⁺ (N-methyl-D-glucamine). The solution was bubbled with 95% O₂/5% CO₂ to maintain the pH of the solution at 7.4.

The epithelium exhibited a basal transepithelial potential difference for every monolayer examined, which was measured with Ag/AgCl reference electrodes (World Precision Instruments, Sarasota, FL) connected to a preamplifier, which was connected in turn to a voltage clamp amplifier (DVC 1000; World Precision Instruments). The change in I_SC was defined as the maximal rise in I_SC following agonist stimulation and was normalized as current per unit area of epithelial monolayer (in µA/cm²). Variations in I_SC between cultures were observed and experiments were normally repeated in different batches of culture to ensure that data were reproducible.

Statistical Analysis

Results are expressed as means ± SEM, n indicates the number of experiments. Comparisons between groups of data were made via Student's unpaired t-test A P value of less than 0.05 was considered statistically significant.

RESULTS

Basal Current Across the Endometrial Epithelium

Under the present culture conditions, the mouse endometrial epithelial cultures exhibited an averaged basal I_SC (I_b) of 24.1 (0.4 µA/cm²; n = 93), and a transepithelial potential of 4.7 ± 0.1 mV, with the apical side negative with respect to the basolateral side. Addition of amiloride at a concentration (10 µM, apical) known to block ENaC inhibited 90.0 ± 1.0% (n = 33) of the basal current, indicating that the I_b was contributed predominately by Na⁺ absorption.

ATP-Induced Inhibition of Na⁺ Absorption

Apical addition of ATP (10 µM) induced a decrease in the I_SC of 2.4 ± 0.3 µA/cm², and subsequent addition of forskolin (10 µM, basolateral) induced a further reduction in I_SC of 1.6 ± 0.3 µA/cm² (n = 13; Fig. 1, A and C), indicating that different inhibitory mechanisms are involved. However, in the presence of the Na⁺ channel blocker, amiloride (10 µM apical), the agonist-induced reduction in I_SC was not observed, but there was an increase of 3.3 ± 0.4 µA/cm² and 6.4 ± 0.8 µA/cm² (n = 8; Fig. 1, B and C) when the I_SC was induced by ATP and forskolin, respectively, which could be inhibited by Cl^- channel blocker, DPC (1 mM; Fig. 1B). The ATP-induced I_SC increase was biphasic and transient, whereas the forskolin-induced I_SC was sustained, results that were similar to those previously reported for the Ca²⁺ and cyclic adenosine monophosphate (cAMP)-activated Cl^- secretions, respectively [4, 6, 7]. A similar ATP and forskolin-induced increase in I_SC, but not decrease, was observed when apical Na⁺ was replaced (data not shown), indicating that the agonist-induced reduction in I_SC was probably due to an inhibition of Na⁺ absorption.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Agonist-induced ISC responses in the absence (A) and presence (B) of amiloride. The horizontal line represents zero ISC and arrows mark the time of drug addition: ATP (10 µM, apical), forskolin (10 µM, basolateral), amiloride (10 µM, apical). C) Mean values of ISC responses to ATP and forskolin corresponding to A and B. Values are means ± SEM. Note that decreases and increases in ISC were observed in the absence and presence of amiloride, respectively

Another possibility for the observed downward response of I_SC is Cl^- absorption. To test this possibility, apical Cl^- concentrations were changed to 100 and 70 mM, and its effect on the ATP-induced I_SC response was examined. As shown in Figure 2A, the ATP-induced reduction in I_SC remained relatively the same for the Cl^- concentrations tested, excluding the involvement of Cl^- absorption. Cl^- channel blockers, DPC and DIDS, were also used to confirm that the endometrial epithelium in basal conditions is not Cl^--absorptive because both blockers resulted in a reduction in the basal I_SC (Fig. 2B; n = 6), which could only be explained by Cl^- secretion in basal conditions. Taken together, the ATP-induced downward I_SC response was indeed due to inhibition of Na⁺ absorption.

View larger version (9K): [in this window] [in a new window]   FIG. 2. Evidence excluding the possible involvement of Cl- absorption. A) Summary of the ATP-induced downward ISC observed when apical Cl- concentration was changed to 100 and 70 mM (n = 4), without significant difference between the two. B) Representative of basal ISC in response to Cl- channel blockers, DPC (2 mM; n = 6) and DIDS (0.1 mM, n = 6)

Involvement of the P₂ Receptors

The stimulatory effect of extracellular ATP on endometrial Cl^- secretion has been shown to be mediated by the P_2U receptor [4]. In the present study, the effect of a P₂ receptor antagonist, reactive blue (100 µM), was tested. Pretreatment of the cells with reactive blue almost completely abolished the ATP-induced inhibition of Na⁺ absorption (n = 6; Fig. 3, A and C), as well as the ATP-stimulated Cl^- secretion (n = 4; Fig. 3, B and D), indicating a possible involvement of the P_2U receptor. On the contrary, both forskolin-induced I_SC responses were not affected (Fig. 3, C and D), further indicating that the ATP-induced responses were unlikely to be mediated by the cAMP-dependent pathway.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Demonstration of involvement of P2 receptor. Agonist-induced ISC responses, under treatment with reactive blue (R. blue), in the absence (A) and presence (B) of amiloride. The horizontal line represents zero ISC and arrows mark the time of drug addition: R. blue (100 µM, apical), ATP (10 µM, apical), forskolin (10 µM, basolateral), and amiloride (10 µM, apical). C) Mean ISC responses to ATP and forskolin without (control) and with R. blue in the absence of amiloride. D) Mean ISC responses to ATP and forskolin without (control) and with R. blue in the presence of amiloride. Note that ISC recordings for the controls are similar to that shown in Figure 1 and, therefore, are not shown in this figure. **Indicates P < 0.01. ***Indicates P < 0.001 when compared with respective controls. The forskolin-induced changes were not significantly affected by R. blue

UDP, which had been shown to selectively activate pyrimidinoceptors, P_2Y4/P_2Y6 [15, 16], was found to induce inhibition of Na⁺ absorption (n = 5), as well as stimulation of Cl^- secretion (n = 4), both of which were distinct from those induced by ATP (Fig. 4), regardless the order of drug addition, indicating that different populations of P₂ receptors are involved.

View larger version (11K): [in this window] [in a new window]   FIG. 4. Additive ISC responses to UDP and ATP in the absence (A) and presence (B) of amiloride. Distinct ISC responses, decreases or increases, to UDP (100 µM, apical), ATP (10 µM, apical), and forskolin (10 µM, basolateral) were observed, indicating the involvement of different receptors or pathways. The order of drug administration did not alter the results. Current recordings are representative of at least three experiments

Ca²⁺ Dependence of the ATP-Induced Inhibition

Both the P_2U receptor and the pyrimidinoceptors are known to be coupled to one or more Ca²⁺-dependent pathways. Experiments were conducted in the present study to confirm whether the extracellular nucleotide-induced inhibition of Na⁺ absorption was Ca²⁺-dependent. The ATP-induced inhibition of Na⁺ absorption, as well as the ATP-stimulated Cl^- secretion, could be mimicked by a Ca²⁺ ionophore, ionomycin (1 µM; n = 5; Fig. 5). A Ca²⁺ chelator, EGTA (2.6 mM, resulting in an extracellular free Ca²⁺ concentration in the nM range), was found to significantly reduce the ATP-induced inhibition of Na⁺ absorption and Cl^- secretion (n = 6), but not the forskolin-induced I_SC (n = 6; Fig. 6), further indicating the involvement of Ca²⁺ in mediating the inhibitory effect of ATP. The intracellular Ca²⁺ chelator, BAPTA-AM, also abolished the ATP-induced reduction in the I_SC (data not shown).

View larger version (11K): [in this window] [in a new window]   FIG. 5. Ionomycin-induced ISC responses in the absence (A) and presence (B) of amiloride. Note that ionomycin (1 µM, apical) mimicked both ATP-induced decrease and increase in ISC. Current recordings are representative of four experiments

View larger version (24K): [in this window] [in a new window]   FIG. 6. Effect of EGTA on ATP-induced responses. Mean values of ISC responses to ATP, without and with EGTA (2.6 mM), in the absence and presence of amiloride (10 µM). **Indicates P < 0.01 when compared with respective controls

Involvement of CaCC

The cAMP-dependent inhibition of ENaC is known to involve the cAMP-activated Cl^- channel, CFTR. The present study tested the possible involvement of the CaCC using DIDS (100 µM), a Cl^- channel blocker known to inhibit this type of Cl^- channel. The ATP-induced inhibition of Na⁺ absorption (n = 7), as well as the ATP-stimulated Cl^- secretion (n = 6), was abolished in the presence of DIDS (Fig. 7), indicating the involvement of Ca²⁺-dependent Cl^- channel.

View larger version (16K): [in this window] [in a new window]   FIG. 7. Involvement of CaCC. Mean current values of ISC responses to ATP, without and with DIDS (100 µM, apical), in the absence and presence of amiloride. DIDS abolished both the ATP-induced decrease and increase in ISC. ***Indicates P < 0.001 when compared with respective controls

Dependence on Cl^- Permeation

Permeation of Cl^- through CFTR is considered critical for the inhibition of ENaC by CFTR [17]. When experiments were conducted in Cl^--free solution, the ATP-induced as well as the forskolin-induced inhibition was reversed (n = 5), indicating that Cl^- permeation was necessary for both cAMP and Ca²⁺-dependent inhibition of Na⁺ absorption (Fig. 8, A and C). The ATP-induced and forskolin-induced increase in I_SC in the presence of amiloride were also significantly reduced (n = 3), which was expected of Cl^- secretion (Fig. 8, B and D). The remainder I_SC observed in the absence of Cl^- was due to the remaining HCO contained in the Cl^--free solution as demonstrated previously [6, 7].

View larger version (19K): [in this window] [in a new window]   FIG. 8. Cl--dependence of the ATP-induced inhibition of Na+ absorption. Agonist-induced ISC responses in Cl--free solution, obtained in the absence (A) and presence (B) of amiloride. C) Mean ISC responses to ATP and forskolin in normal (K-H) and Cl--free solution (on both sides) in the absence of amiloride. D) Mean ISC responses to ATP and forskolin in normal (K-H) and Cl--free solution in the presence of amiloride. Note that ISC recordings for the controls are similar to that shown in Figure 1 and, therefore, are not shown in this figure. See text (Materials and Methods) for details on solutions. ***Indicates P < 0.001. **Indicates P < 0.01. *Indicates P < 0.05 when compared with respective controls

DISCUSSION

In addition to its role in stimulating endometrial Cl^- secretion, which was previously demonstrated [4], ATP has been shown, by the present study, to have an inhibitory effect on Na⁺ absorption. It should be noted that the culture conditions, such as plating cell density and Matrigel coating, were altered in the present study in order to maximize the expression of ENaC so that the basal current, which was predominately contributed by Na⁺ absorption, is more than five times larger than that reported in the previous study [4]. Therefore, inhibition of Na⁺ absorption could be readily observed in the present study but not the previous study as a reduction in the basal current. The influence of culture conditions on the expression of ENaC has been demonstrated previously in airway epithelial cells [18]. The alteration in ENaC expression in the endometrium is of physiological significance because the endometrium is under a constant influence of dynamically changed ovarian hormones. Our recent studies indicate that estrogen and progesterone have different effects on the pattern of ENaC expression (unpublished data). Therefore, in the present culture conditions, the epithelia may represent a physiological state in which ENaC is highly expressed.

The ATP-induced reduction in I_SC is taken as an indication of inhibition of Na⁺ absorption because it was not observed upon removal of apical Na⁺ or in the presence of the ENaC blocker, amiloride. Instead, an increase in I_SC was observed under these conditions with current kinetics that were similar to those previously reported for ATP-induced Cl^- secretion [4]. This suggests that extracellular ATP concurrently stimulates Cl^- secretion (as demonstrated by the upward I_SC in the presence of amiloride) and suppresses Na⁺ absorption (as demonstrated by the downward I_SC in the absence of amiloride). One can argue, however, that the upward I_SC response or Cl^- secretion observed in the presence of amiloride may be due to the strong inhibition of the high-rate Na⁺ influx, which hyperpolarized the apical membrane and changed the direction of the electro-chemical gradient of Cl^- across the apical membrane, changing from Cl^--absorptive in basal conditions to Cl^--secreting in the presence of amiloride. Based on this argument, the ATP-induced reduction in basal I_SC could be interpreted as an increase in Cl^- absorption. However, the present study has provided evidence to rule out this possibility. First, changes in apical Cl^- concentrations did not significantly affect the ATP-induced downward I_SC, whereas Cl^- absorption should have shown a dependence on the apical Cl^- concentrations. Second, both the Cl^- channel blockers, DPC and DIDS, resulted in a reduction in basal I_SC, while inhibition of Cl^- absorption by channel blockers should have given rise to an upward response. Taken together, the present study has demonstrated the inhibition of Na⁺ absorption upon stimulation with ATP, in addition to activation of Cl^- secretion. This provides a mechanism to switch from a predominately Na⁺ absorptive state (basal condition) to one with maximal Cl^- secretion (upon stimulation). The same phenomenon has been observed in rabbit tracheal epithelium [9]. UTP as well as ATP-induced inhibition of Na⁺ transport has also been observed in porcine thyroid [11], rat fetal lung distal epithelial cells [12], and human bronchial epithelia [10]. However, the detailed mechanism underlying the inhibitory effect of ATP has not been fully elucidated.

Although P_2Y2 (P_2U) receptors had been implicated in mediating the inhibitory effect of ATP on Na⁺ absorption in previous studies [11, 12], the present study indicates that the inhibitory effect by extracellular nucleotides is not restricted to P_2Y2 (P_2U) receptors because UDP, which a relatively pyrimidinoceptor-selective agonist [15, 16], could also induce inhibition of Na⁺ absorption distinct from that induced by ATP. The present study has further demonstrated the Ca²⁺ dependence of the extracellular nucleotide-induced inhibition of Na⁺ absorption. The effect of extracellular nucleotides could be mimicked by the Ca²⁺ ionophore, ionomycin, but inhibited by Ca²⁺ chelators, EGTA and BAPTA-AM, indicating that a Ca²⁺-dependent mechanism is involved. In contrast to the study conducted in rabbit tracheal epithleium [9], in which the Ca²⁺ ionophore failed to induce the inhibitory effect and the ATP-induced inhibition was independent of extracellular Ca²⁺, the present study has clearly demonstrated an important role of Ca²⁺ influx in inhibiting Na⁺ absorption in the mouse endometrial epithelium, suggesting that the Ca²⁺-dependent mechanism in the two species may be different. The Ca²⁺-dependence of the inhibitory effect exerted by extracellular nucleotides can be explained by the observed inhibition of ion channels, presumably ENaC, by Ca²⁺ [19, 20]. Basolateral transport mechanisms, such as the Na⁺ pump, have also been shown to be inhibited by Ca²⁺ [21, 22]. The present study does not exclude the possible involvement of protein kinase C (PKC); however, the present results suggest that the possible involvement of PKC may also depend on Ca²⁺ rather than a Ca²⁺-independent mechanism [23, 24].

The Ca²⁺ dependence of the ATP-induced inhibition of Na⁺ absorption is further supported by its dependence on the Ca²⁺-activated Cl^- channels. The presence of DIDS, which is known to inhibit CaCC, abolished the inhibitory effect of ATP, suggesting that the inhibition requires the activation of CaCC. The involvement of Cl^- channels in mediating the inhibitory effect on Na⁺ absorption has been demonstrated for the cAMP-dependent inhibition of ENaC, which requires the activation of CFTR [8]. The present study has further demonstrated the dependence of the ATP-induced inhibition on extracellular Cl^-, which may imply that the permeation of Cl^- through CaCC is necessary. Similarly, Cl^- permeation through CFTR has been demonstrated to be the prerequisite for exerting its inhibitory effect on ENaC [17]. Note that in the present study, Cl^- replacement resulted in the abolishment of both ATP-induced and forskolin-induced reduction in I_SC, which is consistent with the notion that both inhibitory mechanisms require Cl^- permeation through their respective ion channels. It should also be noted that throughout the present study, all experiments testing the involvement of purinoceptors and Ca²⁺ did not significantly alter the forskolin-induced effect, indicating that the ATP-induced Ca²⁺-dependent inhibition of Na⁺ absorption in the mouse endometrial epithelium is independent of the cAMP-dependent mechanism mediated by CFTR. The mechanism as to how activation of CaCC leads to inhibition of ENaC remains to be elucidated.

The present study has indicated that the signaling pathway for the inhibition of Na⁺ absorption by extracellular ATP is similar to that for the stimulation of Cl^- secretion previously reported in the mouse endometrial epithelium [4]. This has the advantage of simultaneous regulation of Cl^- secretion and Na⁺ absorption, giving rise to high efficiency in regulating the uterine fluid microenvironment. The uterine fluid volume is determined by the net electrolyte transport across the endometrium, and stimulated Cl^- secretion with suppressed Na⁺ absorption could result in the maximal driving force for the accumulation of fluid in the uterine lumen, perhaps to facilitate sperm movement. The large amount of ATP contained in the sperm and semen [25, 26] may be released into the uterine tract to elicit secretory activity and suppress absorptive activity. Therefore, extracellular nucleotides may play an important role in the fine-tuning of the uterine fluid microenvironment by regulating both Cl^- secretion and Na⁺ absorption across the endometrium.

ACKNOWLEDGMENTS

We thank Mr. Y.W. Chung for his technical assistance.

FOOTNOTES

First decision: 15 May 2000.

¹ Supported by the Strategic Research Programme and Direct Grant of the Chinese University of Hong Kong.

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

Accepted: August 3, 2000.

Received: April 17, 2000.

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