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Am J Physiol Endocrinol Metab 295: E55-E62, 2008. First published April 22, 2008; doi:10.1152/ajpendo.00714.2007
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Hormonal and purinergic stimulation of bicarbonate secretion in oviducts of rhesus monkey

M. Rajagopal,1 H. Fischer,2 and J. H. Widdicombe1

1Department of Physiology and Membrane Biology, University of California-Davis, Davis; and 2Children's Hospital Oakland Research Institute, Oakland, California

Submitted 8 November 2007 ; accepted in final form 11 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because an increase in the HCO3– concentration of oviductal liquid at midcycle is believed to markedly enhance fertility, we have studied active secretion of HCO3– across highly differentiated cultures of monkey oviductal epithelium. Cultured cell sheets were mounted in Ussing chambers and bathed in medium containing 25 mM HCO3–. Purinergic agents potently stimulated short-circuit current (Isc) with an initial transient response declining within ~2 min to a sustained response. The potency sequence of ATP {approx} UTP > ADP >> AMP suggested that the Isc response was mediated mainly by P2Y2 receptors. Acetazolamide, an inhibitor of carbonic anhydrase, had little or no effect on baseline Isc or the transient response to ATP but abolished the sustained response to ATP. Similar results were obtained on sheets of native epithelium. In pH-stat experiments, the abluminal medium of cell cultures was bathed in HCO3–-CO2 medium, and the pH of the unbuffered luminal medium was maintained at ~7.4 by addition of strong acid or base. ATP stimulated base secretion, and this was inhibited by acetazolamide. Furthermore, these changes in secretion of base were in good quantitative agreement with the Isc responses. When phenol red (an estrogen) was removed from the culture medium, ATP-dependent HCO3– secretion was markedly reduced but could be restored by treatment with estradiol. Estrogens also markedly increased ciliation of the cultures. These results suggest that the midcycle increase in the HCO3– concentration of oviductal liquid may be mediated by the effects of estradiol on purinergic pathways or on ATP secretion.

carbonic anhydrase; short-circuit current; estradiol; adenosine 5'-triphosphate

THE OVIDUCTAL EPITHELIUM regulates the composition of the oviductal liquid, thereby creating a favorable environment for key reproductive events to take place. Among the most important components of oviductal liquid is HCO3–, which has been shown to promote oocyte maturation, sperm capacitation, fertilization, and zygote development (2). In fact, defective HCO3– transport might cause reduced fertility in women with cystic fibrosis (35).

In vivo studies in many different species have shown that the bicarbonate concentration of oviductal liquid peaks in midcycle at approximately the same time as ovulation and a peak in plasma estrogen levels (13, 20, 23). In rhesus monkeys, for instance, Maas et al. (20) measured a midcycle pH change from 7.1–7.3 to 7.5–7.8. From the simultaneously measured values of PCO2, the HCO3– concentration was calculated to increase from 35 to 90 mM (20). Given that oviductal epithelium shows a lumen negative potential difference (5, 18), such a large increase in HCO3– concentration must involve active secretion of HCO3– by the oviductal epithelium.

It is likely that this secretion of HCO3– is by the same mechanism as that proposed for several epithelia, including those of the pancreas and airways (30, 31). This classical mechanism has HCO3– entering the cell from the interstitium via a Na+:HCO3– cotransporter or generated from CO2 by carbonic anhydrase. The bicarbonate ions then exit via the apical membrane through a Cl–:HCO3– exchanger or anion channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). Anion channels also function to recycle the Cl– entering the cell by the Cl–:HCO3– transporter (31).

However, further analysis of either cellular mechanisms or of the neurohumoral regulation of HCO3– secretion by oviductal epithelium would be facilitated by measurements of net transfer of HCO3– across isolated sheets of epithelium in vitro. Unfortunately, studies of native epithelium are made difficult by a number of factors including limited availability, dissection trauma, loss of viability with time after removal from the animal, nonsterile conditions, the smallness and cylindrical nature of the oviduct, and the presence of submucosal tissues. Finally, the highly plicated nature of native epithelium precludes accurate measurements of epithelial surface area. These problems can all be circumvented by the use of cell cultures. Thus pure preparations of oviductal epithelial cells can be cultured as planar sheets of known surface area (generally ~1 cm2). The starting material can be expanded. Cells may remain viable over a long period in culture. Individual cultured sheets can be studied immediately on removal from the incubator. Finally, sterile conditions allow prolonged exposures to neurohumoral agents.

Of course, the major problem with cell culture is that cultured cells may be dedifferentiated compared with native epithelium. However, on the basis of culture approaches developed by us for airway epithelium (34), our laboratory has recently produced highly differentiated cultures of monkey oviductal epithelium (26). These cultures become densely ciliated and contain mature secretory cells resembling those of native epithelium. High transepithelial electrical resistance (Rte) indicates that tight junctions have formed between the individual cells. Finally, the presence of a lumen negative transepithelial potential difference (Vte) indicates that the cells have polarized, contain distinct apical and basolateral membranes, and show vectorial transepithelial transport of ions. When mounted in Ussing chambers, Vte can be clamped to zero by current flow in an external circuit. This short-circuit current (Isc) is equal to the sum of the active ion transport processes operating across the epithelium (32). As reviewed in detail earlier (26), our cultures have Rte and Isc very similar to that of native epithelium. Furthermore, the main active ion transport process operating across our cultures under baseline conditions is secretion of Cl–, again in good agreement with results on native epithelium (5, 18). Thus, both structurally and functionally, our cultures are a good model for native epithelium.

Being in the form of planar cell sheets on porous supports, the cultures are ideal for studying transepithelial transport processes. We have earlier shown that luminal ATP is a potent stimulator of Isc across these cultures (26). These results were obtained in the presence of amiloride to inhibit active absorption of Na+, so the ATP-dependent increase in Isc presumably represents anion secretion. Here we show that a major component of this increase in Isc is active secretion of HCO3–. We further show that this ATP-dependent secretion of HCO3– is markedly enhanced by estrogens. Importantly, we obtained similar results on native epithelium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oviducts from rhesus monkeys (Macaca mulatta) were obtained from the California National Primate Center as part of a tissue-sharing program. On removal from the animal, they were immediately placed in ice-cold saline and generally arrived at our laboratory within 2 h of removal. Cells were obtained by protease digestion as previously described (26). Initial yields averaged 9 ± 2 x 106 cells per pair of oviducts (n = 28). Eight suspensions of dispersed cells were plated at 106 cells/cm2 onto 12-mm transwell polycarbonate inserts (0.4-µm pore size; Costar, Corning, NY) or Snapwell polycarbonate inserts (Costar) that were coated with human placental collagen (Sigma-Aldrich, St. Louis, MO) as described previously (7). Of the remaining 20 isolates, eight that yielded less than 4 million cells were plated in single T-25s, while others with higher yields were plated in T-75s at a density of 4–7 x 106 cells per flask. Once the cells in a flask were 90% confluent, they were trypsinized and plated onto inserts at 106 cells/cm2. Yields after expansion averaged 16 ± 3 million cells per pair of oviducts, an increase of approximately threefold over the initial yield. Plating of cells onto flasks or inserts was in a 1:1 mixture of Dulbecco's minimum essential medium (DMEM) and Ham's F-12 medium (F12) supplemented with 5% FCS. The day after plating, the culture medium was changed to "Gray's medium" (12, 28). This medium consists of a 1:1 mixture of DMEM and "bronchial epithelial basal medium" supplemented with the following: hydrocortisone (1.4 µM), transferrin (10 µg/ml), epidermal growth factor (0.5 ng/ml), insulin (5 µg/ml), epinephrine (2.7 µM), triiodothyronine (9.7 nM), all-trans retinoic acid (50 nM), bovine serum albumin (1.5 µg/ml), and bovine pituitary extract (1%). Phenol red was present in both stock media to produce a final concentration of 23 µM. However, for studies on the effects of estradiol or progesterone, we used a medium containing the same growth supplements as Gray's medium but with a base consisting of a 1:1 mixture of DMEM and F12, both free of phenol red. All media contained penicillin-streptomycin (100 U/ml), gentamycin (100 µg/ml), and fungizone (2.5 µg/ml). The sources of our cell culture media components are given elsewhere (28).

Approximately daily after the plating onto inserts, transepithelial resistance (Rte) was determined with a chopstick voltmeter (Millicell-ERS; Millipore, Billerica, MA). Cell sheets were deemed usable once Rte became ≥200 {Omega}·cm2. This occurred from 3 to 5 days after plating, and the age of the cells was referenced to this point. Thus, when the age of the cells is given in the text and figure legends, it refers to the days after achieving Rte ≥200 {Omega}·cm2, not to days after plating. Once cell sheets attained the acceptable Rte, they were studied in conventional Ussing chambers (exposed area 0.5 cm2) in Krebs-Henseleit solution (in mM: 140 NaCl, 25 NaHCO3, 5 KCl, 5 glucose, 2 CaCl2, and 1 MgCl2) and gassed with 95% oxygen and 5% CO2 to produce a pH of 7.4. Vte was clamped to 0 mV, and the resulting Isc was displayed continuously on a chart recorder. A set voltage pulse (0.5–2.0 mV) was applied across the cell sheet for 200 ms every 20 s, and Rte was determined from the ratio of applied voltage to additional clamp current required. Rte determined in Ussing chambers was ~40% of that measured with the chopstick voltmeter, a result we have also seen with tracheal epithelial cell cultures (unpublished data). Possible reasons for this include edge damage in the Ussing chambers as well as differences in geometry and medium circulation between the two measuring systems. Isc and Rte stabilized within the first 5 min of mounting of the tissue, at which point a series of pharmacological agents (from Sigma-Aldrich unless otherwise stated) were added. All tissues were first treated with 10–5 M amiloride in the luminal medium to inhibit Na+ absorption. Then 10–5 M forskolin (MP Biomedicals, Aurora, OH), was usually added to both sides of the cell sheet to stimulate cAMP-mediated Cl– transport. Next, purinergic agents (10–5 M to both sides unless otherwise stated) were added to stimulate Ca2+-dependent anion secretion. In some experiments, this was followed by addition of 2 x 10–4 M acetazolamide, an inhibitor of carbonic anhydrase, to both sides. In four cell sheets, 10–5 M 4,4'-diisothiocyano-2, 2'-stilbenedisulfonate (DIDS) was added to the abluminal side to block Na+:HCO3– cotransport. Finally, 10–3 M Cl– channel blocker, diphenylamine-2-carboxylate (DPAC), was added to both sides. All agents were added as 1:100 dilutions of stocks in ethanol, dimethyl sulfoxide, or water. Vehicle controls had no effect on Isc. When cells were treated with estradiol or progesterone, these hormones were added as soon as cells achieved Rte ≥200 {Omega}·cm2. As in earlier studies, there were no significant electrophysiological differences between cells plated directly onto inserts or cells first expanded in tissue culture flasks (26).

To study ion transport across native epithelium, sections of oviductal wall from rhesus monkeys were mounted in conventional Ussing chambers as quickly as possible following removal from the animal (generally within 2 h). The Ussing chambers had an aperture of 0.125 cm2, and solution resistance was too high to be compensated for by the voltage clamp apparatus. Accordingly, in these experiments, the open-circuit Vte was continuously recorded, and voltage changes in response to small current pulses (1–5 µA every 20 s) were measured and total resistance determined. At the end of the experiment, tissues were removed and solution resistance was determined. Rte was then taken as the difference between total and solution resistance, and Isc was calculated at the time of every current pulse from the ratio of Vte to Rte. Viability of native epithelium was assessed in two ways: first from the Isc response to circulation of warm oxygenated Krebs-Henseleit solution, and second from the effects of the Cl channel blocker, DPAC, added at the end of the experiment. Tissues were mounted in Ussing chambers that were then connected to the gas-lift oxygenators. Sufficient Krebs-Henseleit solution was added just to cover the faces of the tissue, and measurements of Isc and Rte were initiated. Then, a minute later, syringes were used to push Krebs-Henseleit solution into the bottom of the chambers and up the tubes connecting the chambers to the reservoirs of the water-jacketed gas-lift oxygenators. As soon as the liquid reached the reservoirs, it was warmed and began circulating across the faces of the tissues, which, if viable, showed an immediate increase in Isc (and decrease in Rte). Results were only used from tissues in which fluid circulation stimulated, and DPAC inhibited, Isc by >5 µA/cm2; only tissues studied within 1 h of removal from the animal met these criteria.

Bicarbonate secretion was measured by pH-stat as described (10). Cell sheets on Snapwells were mounted in a commercially available Ussing chamber (Physiologic Instruments, San Diego, CA), and abluminal and luminal surfaces were bathed with 5 ml of media and maintained at 37°C. The abluminal surface was bathed in the Krebs-Henseleit solution used in our conventional Ussing chamber studies. The luminal side was bathed in Krebs-Henseleit solution in which the NaHCO3 had been replaced by an equivalent amount of NaCl. Immediately before use, this luminal solution was titrated to the desired pH of 7.4 using 10 mM NaOH. The luminal side was oxygenated with 100% O2. Tissues were short-circuited and Rte and Isc recorded. Luminal pH was monitored continuously and manually titrated to a target value of 7.4 with 10 mM HCl as described (10). From the added volume of HCl and the time between additions, HCO3– fluxes, in µeq·cm–2·h–1, were calculated. Stock solutions of pharmacological agents were adjusted to pH 7.4 before addition.

Scanning electron microscopy was as described previously by our laboratory (26).

Tests for statistically significant differences between means were done with Student's t-test (paired or unpaired), with P < 0.05 being regarded as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Twenty-eight cultures were initiated. Even after several weeks, six of these failed to develop resistance >200 {Omega}·cm2 and were discarded. However, twenty-two cultures developed an acceptable Rte of ≥200 {Omega}·cm2 between 3 and 5 days after plating and were used in further studies.

In four cultures (31 cell sheets), Rte, Isc, and responses of Isc to pharmacological agents were measured in Ussing chambers from 3 to 20 days after the development of Rte >200 {Omega}·cm2 as measured with the "chopstick" voltmeter. Least-squared linear regressions showed no dependence of either Rte or Isc on age of culture (R = 0.105 and 0.026, respectively). For all cultures, cell sheets aged 20 days or less had Rte of 115 ± 12 {Omega}·cm2 and baseline Isc of 19.7 ± 2.2 µA/cm2 (n = 93). Amiloride and forskolin were added sequentially to all cultures to inhibit active Na+ absorption and stimulate cAMP-dependent Cl– secretion, respectively. Amiloride produced a small inhibition of Isc (1.7 ± 0.3 µA/cm2), and forskolin produced a small stimulation (3.4 ± 0.5 µA/cm2). The responses to amiloride and forskolin were not age dependent (R = 0.031 and 0.230, respectively; best least-squared linear regressions).

As described elsewhere (26), ATP added to both sides of the tissue after amiloride and forskolin potently stimulated Isc, with a transient increase being followed in ~2 min by a lesser sustained response. By contrast to the responses to amiloride and forskolin, the responses to ATP were significantly age dependent (R = 0.496 for the transient response, and 0. 484 for the sustained; best least-squared linear regressions; n = 31). Between 0 and 5 days after attainment of acceptable Rte, ATP had no significant effect on Isc. Between days 6 and 10, ATP produced a transient response of 22.0 ± 5.0 µA/cm2 and a sustained response of 7.0 ± 2.0 µA/cm2. Between 11 and 20 days, the transient response averaged 41.0 ± 6.0 µA/cm2 and the sustained 14.0 ± 3.0 µA/cm2 (Fig. 1). These data suggest a correlation between the transient and sustained responses to ATP. This was confirmed by best least-squared regression analysis for individual cell sheets, which showed a highly significant correlation (R = 0.795; n = 31) between the sustained and transient responses for cultures between 3 and 20 days after attaining Rte ≥200 {Omega}·cm2. After day 20, there was a decline in baseline Isc and the responsiveness to ATP (data not shown). Therefore, in the results presented below, tissues were studied between 10 and 20 days of attaining acceptable Rte.


Figure 1
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  		Fig. 1. Dependence of short-circuit current (Isc) increase in response to ATP on age of culture. Isc was measured in conventional Ussing chambers. ATP (10–5 M) added to both sides of cultured cell sheets induced a transient increase in Isc ({square}) that peaked after 5 s and then declined within ~2 min to a stable sustained level ({blacksquare}). Data from cultures aged 0–5, 6–10, or 11–20 days (d) were pooled. Both transient and sustained responses increased significantly with each increase in age interval. Values are means ± SE; n = 5–16.

 
To determine whether ATP was stimulating HCO3– secretion, we tested whether the increase in Isc in response to ATP could be inhibited by blockers of carbonic anhydrase or blockers of Na+:HCO3– cotransport. We found that acetazolamide, an inhibitor of carbonic anhydrase, produced a small inhibition of Isc (–2.3 ± 0.7 µA/cm2) if given before ATP, but after ATP it produced a significantly greater inhibition of –11.8 ± 3.7 µA/cm2 (Fig. 2, A–C). Furthermore, pretreatment with acetazolamide significantly inhibited the sustained response to ATP (Fig. 2, A, B, and D).


Figure 2
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  		Fig. 2. Inhibitory effects of acetazolamide on Isc response to ATP. Pretreatment with acetazolamide inhibited the Isc responses of cultured cell sheets to ATP. Conversely, ATP induced sensitivity to acetazolamide. A: typical record. A small stimulation by 10–5 M forskolin (F) was followed by a larger stimulation by 10–5 M ATP (ATP) and a marked inhibition by 2 x 10–4 M acetazolamide (Ac). B: typical record showing that acetazolamide had no effect on baseline Isc but blocked the sustained increase in Isc in response to ATP. Cell sheet from the same culture as in A. C: effects of acetazolamide of Isc. Given after ATP, acetazolamide produced a much larger inhibition of Isc than when given before ATP. Values are means ± SE of 9 pairs of cultured cell sheets. *Significantly different from value obtained when added before ATP. D: effects of acetazolamide on response to ATP. Pretreatment with acetazolamide significantly inhibited the sustained response to ATP ({blacksquare}) but was without effect on the transient response ({square}). Values are means ± SE of 9 pairs of cultured cell sheets. *Significantly different from value obtained in the absence of acetazolamide. A–D: cultures used were 12–16 days old.

 
An inhibitor of Na+:HCO3– cotransport, DIDS, added after acetazolamide, had complex effects on Isc (n = 4), producing a transient (~30 s) stimulation (+16.6 ± 3.7 µA/cm2) followed by a transient (~30 s) inhibition (–7.6 ± 3.2 µA/cm2), after which Isc stabilized to a value 2.0 ± 1.4 µA/cm2 above the preexposure value.

In virtually all experiments, ATP was added to both sides of the tissue. However, in paired tissues from three cultures, we found that ATP increased Isc more potently from the luminal than the abluminal side (Fig. 3).


Figure 3
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  		Fig. 3. Sidedness of action of ATP on Isc. ATP (10–5 M) was added to either the luminal or abluminal sides of the 12- to 15-day-old cultures. Transient ({square}) and sustained responses ({blacksquare}) to ATP were significantly greater when this agent was added to the luminal than to the abluminal side. Values are means ± SE for 3 pairs of cell sheets, each from a different culture. *Significantly different from corresponding value for luminal addition (paired t-test).

 
The response to ATP was not influenced by pretreatment with forskolin. The transient and sustained responses to ATP were 31.0 ± 7.8 and 12.7 ± 1.3 µA/cm2 in the presence of forskolin, respectively, and 30.3 ± 7.2 µA/cm2 and 10.7 ± 5.2 µA/cm2 in the absence (n = 3 pairs of tissues).

The maximal response to ATP was greater than to UTP or ADP (Fig. 4). The dissociation constants (Kds) for ATP and ADP were constant between cultures at ~10–7 and 10–6 M, respectively. By contrast, the Kd for UTP was highly variable, ranging from 10–8 to 10–6 M. AMP and adenosine were without effect on Isc at doses up to 10–4 M.


Figure 4
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  		Fig. 4. Cumulative dose responses to purinergic agents. Cumulative doses of UTP ({blacktriangleup}), ATP (bullet), or ADP ({blacksquare}) were added to cultured cell sheets in Ussing chambers, and the steady-state values of Isc were recorded. Data are for single cell sheets from the same culture at 12 days of age. Other cell sheets from this culture showed no Isc responses to AMP or adenosine, although they did respond to ATP added at the end of the experiment. Nucleotides were added simultaneously to both sides of the cell sheets. Similar results were obtained for UTP, ATP, ADP, AMP, and adenosine on 3 other cultures.

 
In all pH-stat experiments, the luminal medium alkalinized under baseline conditions, and whenever pH reached 7.5, 5 µl of 10 mM HCl were added. The rate of alkalinization was then calculated from the frequency of addition of the acid aliquots. Figure 5 shows a typical result, in which base secretion was little affected by forskolin but was markedly stimulated by ATP. In 11 experiments, ATP increased base secretion from 0.32 ± 0.04 to 0.83 ± 0.21 µeq·cm2·h–1 (P < 0.05; paired t-test). Added after ATP, acetazolamide significantly (paired t-test) inhibited base secretion from 0.42 ± 0.19 to 0.15 ± 0.07 µeq·cm2·h–1 (n = 4).


Figure 5
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  		Fig. 5. Stimulation of base secretion by ATP. Top: 15-day-old cultured cell sheet was mounted in an Ussing chamber and exposed to HCO3–-CO2 medium (pH 7.4) on its abluminal surface, while the pH of the unbuffered luminal medium was continuously recorded. Luminal medium spontaneously alkalinized, and each time pH reached 7.5, HCl (5 µl of 10 mM) was added as indicated by the vertical lines. Addition of ATP (10–5 M) at "A" markedly increased the rate of alkalinization, whereas forskolin (10–5 M) added at "F" was comparatively ineffective. Bottom: rates of base secretion calculated from the frequency of addition of acid.

 
The above experiments were all performed in medium containing phenol red, which is an estrogen (1). Omission of phenol red from the medium virtually abolished the induction of acetazolamide-sensitive Isc by ATP (Fig. 6). However, treatment with estradiol (500 pM for 14 days) restored sensitivity to both ATP and acetazolamide (Fig. 6). By contrast, progesterone (500 pM for 14 days) did not (data not shown).


Figure 6
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  		Fig. 6. Effects of estradiol on Isc responses to ATP and acetazolamide. Cultured cell sheets were exposed to estradiol (500 pM) for 14 days and then mounted in conventional Ussing chambers and exposed to ATP (10–5 M), followed by acetazolamide (2 x 10–4 M). Control cell sheets from the same cultures were studied after identical periods in culture but were not exposed to estradiol. The transient (t) and sustained (s) increases in response to ATP are shown together with the inhibition by acetazolamide for control ({square}) and estradiol-treated ({blacksquare}) cell sheets. Values are means ± SE; n = 7 tissue pairs. In all cases, the responses of estradiol-treated cells were significantly different from control.

 
We questioned whether the increased responsiveness to ATP with age of the cultures reflected dedifferentiation over time. However, similar to tracheal cultures (17, 33), differentiation increased over the first 20 days in culture, as reflected by a progressive increase in the degree of ciliation. Thus there were no cilia on cultures that were 0–5 days old, but ciliation increased to 40–60% of individual apical membranes on 15-day-old cultures (Fig. 7, A and B). Furthermore, treatment with estradiol for 14 days in medium lacking phenol red was also associated with a marked increase in degree of ciliation of the cell cultures. On average, 60% of individual membranes of estradiol-treated filters were ciliated compared with the 10–20% of untreated time controls (Fig. 7, C and D). Numbers of cilia per ciliated cell were also increased by estradiol. In some instances, estradiol also induced the development of mature secretory cells with protruding apices (Fig. 7D), a change shown previously also to be induced with phenol red (26).


Figure 7
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  		Fig. 7. Estrogen-dependent increase in ciliation. Scanning electron micrographs of the apical surface of cultured cell sheets. Cultures grown in medium containing phenol red had no cilia at 5 days of age (A) but became densely ciliated by 15 days (B). Cultures grown in medium that lacked phenol red failed to develop cilia even 14 days after attaining transepithelial electrical resistance (Rte) ≥200 {Omega}·cm2 (C). However, cells grown in medium lacking phenol red but exposed to estradiol (500 pM) for 14 days not only became densely ciliated but also developed mature secretory cells with protruding apices (D). Cells in A and B are from one culture; cells in C and D are from another. Similar results were obtained on at least 3 other cultures for both phenol red and estradiol. Scale bar = 10 µM for A–C and 20 µM for D.

 
Thus increases in the level of ultrastructural differentiation of our cultures were associated with increased responsiveness to ATP. This suggested that the ATP response was not an artifact of culture but should be present in native epithelium. To test this, three sheets of oviductal wall from rhesus monkeys were mounted in Ussing chambers sufficiently soon after removal from the animals to meet the criteria of viability detailed in MATERIALS AND METHODS. All showed Isc responses to ATP that were considerably greater than to forskolin. Furthermore, acetazolamide added after ATP potently inhibited Isc (Fig. 8). Thus native epithelium gave results that were both qualitatively and quantitatively in excellent agreement with the results in cultured cells (Fig. 8). In both cultured and native epithelium, the Cl– channel blocker DPAC, added at the end of the experiment, essentially abolished Isc (Fig. 8).


Figure 8
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  		Fig. 8. Comparison of Isc responses of native and cultured epithelium. Top: cultured cell sheet exposed sequentially to 10–5 M forskolin (F), 10–5 M ATP (ATP), 2 x 10–4 M acetazolamide (Ac), and 10–3 M diphenylamine-2-carboxylate (D). Bottom: sheet of native epithelium exposed to the same sequence of reagents at the same concentrations. Note the qualitative similarity in responses of native and cultured epithelium. Note also that the Cl– channel blocker, DPAC, added at the end of the experiment, essentially abolishes Isc. Similar records were obtained on 2 further sheets of native tissue and multiple sheets of cultured cells. The break in the trace for cultured cells is 2 min.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, by measuring transfer of both charge and base equivalents across isolated sheets of epithelium, we have confirmed that monkey oviductal epithelium actively secretes HCO3–. However, novel findings of our studies are that this HCO3– secretion is induced by either ATP or UTP from the luminal (but not the abluminal) side of the tissue, and that this nucleotide-dependent secretion of HCO3– is not seen unless tissues have been primed with estrogens. Importantly, similar results were obtained on both cultured and native epithelium. This validates the use of cultures of oviductal epithelium for studies of transepithelial transport in general and HCO3– secretion in particular. It also suggests that in vivo increased secretion of purinergic agents or enhancement of purinergic signaling pathways may be involved in the midcycle increase in HCO3– concentration of oviductal liquid.

Measurements of pH and PCO2 of monkey oviductal liquid in vivo have shown that its HCO3– concentration increases significantly from ~35 to ~75 mM at midcycle (20). In these experiments, PCO2 of liquid from individual oviducts was quite variable, ranging from 46 to 143 Torr, but showed no dependence on the stage of the cycle (20). Therefore, given that active proton absorption has yet to be described for mammalian epithelia, and given also the luminal potential difference of the oviduct is negative (5, 18), it is virtually certain that the midcycle increase in HCO3– concentration is due to active secretion of HCO3–. Thus our finding of active HCO3– secretion across cultured and native oviductal epithelium is confirmatory. However, the ability to study this process in well-differentiated cultures permits detailed analysis of the molecular and regulatory processes involved.

In particular, we here show that HCO3– secretion is absent from the cell cultures unless they are both primed with estrogens and treated with ATP (or UTP) from the luminal side. We were worried that this ATP dependence was an artifact of culture, perhaps reflecting dedifferentiation of the cells. However, sheets of native epithelium showed responses to ATP, acetazolamide, and the other pharmacological agents that were very similar to those of the mature differentiated cultures. Furthermore, responsiveness of cell cultures to ATP was only seen under conditions that permitted differentiation of the cultures as revealed by a marked increase in their degree of ciliation.

Our initial studies were done in commercially available culture medium containing phenol red, an estrogen (1). In this medium, levels of ATP-dependent HCO3– secretion increased progressively over the first 20 days in culture (Fig. 1), a change that paralleled a progressive increase in the degree of ciliation (Fig. 7, A and B). When phenol red was omitted, then the cultures failed to become ciliated (Fig. 7C) and also failed to develop ATP-dependent HCO3– secretion (Fig. 6). However, if estradiol was added to medium lacking phenol red, not only did the ATP-dependent HCO3– secretion return (Fig. 6), but the cultures also increased their degree of ciliation (Fig. 7D). Finally, in suboptimal medium (DMEM-F-12 supplemented with 5% FCS), cell sheets were highly dedifferentiated and only ~5 µm in height (data not shown) as opposed to ~20 µm in Gray's medium without estrogens and ~40 µm in Gray's medium with estrogens. Cells lacked cilia in FCS-supplemented medium, the Isc response to ATP was greatly attenuated, and acetazolamide had no effect on Isc (data not shown).

In native epithelium, the degree of ciliation is greatest in midcycle when estrogen levels are at their peak (3, 24). Furthermore, estrogens also increase ciliation and cell height of native epithelium in spayed monkeys (4, 24). The fact that estrogens had the same ultrastructural effects on our cultures as they have on native epithelium increases our confidence that estrogens induce ATP-dependent HCO3– secretion in native as well as cultured epithelium.

ATP and UTP released across the apical membrane act as potent autocrine agents affecting a variety of functions in many epithelia (29). Of particular relevance to the present report, ATP has been reported transiently to stimulate Isc or Vte across cultures of bovine, mouse, and human oviductal epithelium (8, 9, 19, 21), and we have earlier shown that ATP stimulates Isc across cultures of monkey oviductal epithelium with not only a transient but also a sustained response (26). Conversion of CO2 to HCO3– by carbonic anhydrase constitutes an important step in HCO3– secretion across many epithelia (6, 15, 16, 31), and we therefore tested the effects of acetazolamide on the response to ATP. We found that acetazolamide blocked or abolished the sustained increase in Isc in response to luminal ATP, and we conclude that this response represents predominantly HCO3– secretion. By contrast, the transient response was not significantly affected by pretreatment with acetazolamide and presumably is due mainly to Cl– secretion. However, in some instances (e.g., Fig. 5), we did see transient stimulation of base secretion by ATP.

In addition to the HCO3– generated from CO2, another source of the HCO3– secreted across the apical membrane is Na+:HCO3– cotransport across the basolateral membrane. The relative importance of these two sources of HCO3– varies between epithelia. However, we found that DIDS, an inhibitor of Na+:HCO3– cotransport, had no sustained effect on Isc. Therefore, we conclude that the bulk of the HCO3– secreted by oviductal epithelium is generated by the action of carbonic anhydrase on CO2 and that the acetazolamide-inhibitable Isc is an accurate measure of active HCO3– secretion by this tissue.

To confirm that ATP was stimulating HCO3– secretion, we measured transfer of acid/base across cultured cell sheets using the pH-stat technique, as adopted previously by our laboratory on studies of airway epithelium (10). In this earlier study, we measured acid rather than base secretion. Also, autotitration is used more frequently than the manual titration used in the present studies. However, manual pH-stat of base secretion, exactly as performed here, has recently been successfully used to demonstrate Ca-dependent stimulation of bicarbonate secretion across Calu-3 cells, an airway epithelial cell line (16). Using pH-stat, we found that cultures showed baseline secretion of base, perhaps reflecting diffusion of HCO3– down its concentration gradient from 25 mM in the basolateral medium to 0 mM in the luminal. ATP, however, resulted in a steady-state increase in base transfer of 0.51 ± 0.23 µeq·cm2·h–1. Importantly, this corresponds to 14 ± 6 µA/cm2, a value that is not significantly different from the sustained increase in Isc of 14 ± 3 µA/cm2. Similarly, when added after ATP, acetazolamide inhibited base transfer by 0.27 ± 0.06 µeq·cm2·h–1, or 7.2 ± 1.6 µA/cm2, again not significantly different from its effects on Isc of –11.8 ± 3.7 µA/cm2. Thus both the Ussing chamber and pH-stat experiments indicate that ATP stimulates secretion of HCO3–, and the two techniques are in good quantitative agreement.

The potency sequence of ATP {approx} UTP > ADP >> AMP suggests that the Isc response was mediated mainly by P2Y2 receptors, a conclusion consistent with findings on the regulation of ciliary beat frequency in the oviduct (22). However, although the Kds for ATP and ADP varied little from culture to culture, the Kd for UTP ranged from 10–9 to 10–6 M. This variability suggests that other receptors, more sensitive to UTP than ATP, may also be present in the oviductal epithelium, and the numbers of these receptors relative to P2Y2 receptors may vary between cultures. Specific receptors that would qualify include the P2Y4 and P2Y6 receptors (27). The presence of multiple receptors all contributing in varying degrees to the end response is consistent with studies on Cl– secretion across tracheal epithelium, in which the main receptor appears to be P2Y2, but a variety of other P2Y receptors have been implicated (29).

The marked inhibition of Isc by DPAC suggests that most of the baseline Isc across our cultures of monkey oviductal epithelium is due to Cl– secretion, in agreement with results on native rabbit oviductal epithelium (5). CFTR is the major Cl– channel in the apical membranes of many secretory epithelia and is activated by cAMP (11). So the finding that forskolin had minor effects on baseline Isc suggests either that CFTR is substantially activated under baseline conditions or that most Isc is carried by channels other than CFTR. Apical membrane anion channels, such as CFTR, are required for sustained HCO3– secretion for two reasons. First, they allow the Cl– that enters by Cl–:HCO3– exchange to recycle across the apical membrane. Second, they may themselves carry a significant amount of HCO3– current. CFTR, for instance, has a bicarbonate permeability that is ~20% of the permeability to Cl– (14, 25). The finding that forskolin did not significantly affect the Isc response to ATP therefore suggests either that CFTR function is not rate limiting or that ATP is activating Ca-dependent Cl– channels. This second possibility is supported by the finding that ATP stimulated Isc to identical degrees in cultures of oviductal epithelium from both wild-type and CFTR knockout mice (19).

Native epithelium was not studied extensively because of difficulties in getting oviducts to the laboratory sufficiently soon after removal from the animals. However, the three tissues deemed viable all behaved as for well-differentiated cultured cells: baseline Isc was mainly due to Cl secretion, ATP gave much larger increases in Isc than forskolin, and acetazolamide added after ATP was inhibitory.

In conclusion, we have shown that luminal ATP stimulates HCO3– secretion across oviductal epithelium. Furthermore, estrogens enhance this action of ATP. In future work, therefore, we will test the hypothesis that the midcycle surge in the HCO3– concentration of oviductal liquid is due to a potentiating effect of estradiol on ATP secretion or on purinergic signaling pathways.


   ACKNOWLEDGMENTS
 
We thank Lorne Sachs for support and guidance and Marrah Lachowicz for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Widdicombe, Dept. of Physiology and Membrane Biology, Univ. of California-Davis, Davis, CA 95616-8664 (e-mail: jhwiddicombe{at}ucdavis.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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