Retinoids modulate P2U purinergic receptor-mediated changes in transcervical paracellular permeability

George I. Gorodeski1,2, Dipika Pal1, Ellen A. Rorke3, Richard L. Eckert2, and Peter Burfeind1

Departments of 1 Reproductive Biology, 2 Physiology and Biophysics and 3 Environmental Health Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In human cervical cells, extracellular ATP induces an acute decrease in the resistance of the lateral intercellular space, the phase I response, followed by a delayed increase in tight junctional resistance, the phase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium (RFM) abolished both responses. Treatment with retinoic acid restored the phase I response in full, but the amplitude of the phase II response was restored only partly. Shorter incubations and lower concentrations of retinoic acid [half-maximal effective concentration (K1/2) = 0.1 µM] were required for restoring the phase I response than were required for reversing the phase II response (K1/2 = 1 µM). The phase I response could be restored by ligands that bind to either retinoic acid receptors (RARs) or retinoid X receptors, but only RAR agonists had an effect on phase II response. RFM had no effect on decreases in resistance induced by ionomycin, but it attenuated phase II-like increases in resistance induced by KCl or by 1,2-dioctanoyl-sn-diglycerol (diC8). Actinomycin D blocked phase II response but not phase I response or the responses to ionomycin, KCl, or diC8. These results suggest that retinoids act on cervical cells via distinct retinoid receptor mechanisms and modulate phase I and phase II changes in resistance by regulating distinct signal mechanisms.

human; cervical cells; extracellular ATP; tight junctions; vitamin A; lateral intercellular space; transepithelial transport; cervical mucus

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE MAIN FUNCTION OF THE epithelium that covers the woman's endocervix is to control reproduction. This function depends on well-regulated secretion of fluid and solutes (the cervical mucus) from the epithelial cells that line the cervical lumen. Recently, we described an in vitro model of human cervical epithelial cells on filters that retain phenotypic characteristics of the endocervical epithelium, thus providing an appropriate in vitro model for the study of transcervical transport (20). The cultured cervical epithelium is also characterized by a relatively high degree of permeability compared with other, more occlusive epithelia (13, 19, 24).

More recent studies suggest that the permeability of the cultured cervical epithelium can be regulated. For instance, extracellular purines (ATP) or pyrimidines (UTP) at micromolar concentrations modulate the paracellular resistance acutely and reversibly (17). ATP stimulates two distinct effects that differ in their time course, signaling, and direction: an acute decrease in resistance (termed phase I response), which is mediated by calcium mobilization (18), acute decrease in cell volume, and subsequently a decrease in the resistance of the lateral intercellular space (RLIS) (13, 17). ATP also stimulates a delayed increase in paracellular resistance (termed phase II response), which is sensitive to pertussis toxin and is mediated by augmented calcium influx, activation of protein kinase C, and an increase in tight junctional resistance (RTJ) (17-19). The net effect on transcervical permeability is the composite of the two effects, and the measured changes in permeability are characterized by a transient decrease followed by a sustained increase in resistance (Ref. 18; see Fig. 1A).

Both effects of ATP were shown to be the result of activation of P2U receptors (17). On the basis of pharmacological criteria, it was suggested that phase I and phase II responses are mediated by two distinct P2U receptors. However, the possibility that the signals diverge downstream from a single receptor has not been ruled out. These novel data are relevant to the regulation of cervical mucus in vivo because epithelial cells express P2U purinergic receptors (7, 15, 17, 22, 23), and ATP can accumulate in the extracellular space at micromolar concentrations (4, 26), which are sufficient to activate the P2U receptor mechanisms.

Vitamin A and related compounds, the retinoids, regulate the reproductive tract epithelia in females (9, 10, 14-16, 21). Vitamin A deficiency results in dryness of mucus membranes (9) and may lead to squamous metaplasia and keratinization of the simple columnar epithelium of the endocervix (10). Recent in vitro studies showed that retinoids regulate the paracellular permeability across the cultured human cervical epithelium (14). The objective of the present study was to study the effect of retinoids on the changes in paracellular resistance induced by the P2U receptor pathways. Our results show that the phase I and phase II responses are differentially regulated by retinoic acid receptor (RAR)- vs. retinoid X receptor (RXR)-selective retinoids.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell Culture Techniques

Experiments were done on CaSki cells, which retain phenotypic characteristics of human endocervical cells (20). CaSki cells form a confluent and polarized epithelium on filters, and the cells express tight junctions that occlude effectively the intercellular space (13, 17-20). The growth of CaSki cells on filters depends on the mode of plating: when plated at low density, cells will reach confluency within 1-2 wk and will form a monolayer; when plated at high density, e.g., as for present study, cells will remain attached on the filter as a bi- and/or trilayer (20). In the latter case, the basal layer contains a confluent layer of vital cells that determines the permeability properties of the epithelium (19, 20). The cells in the superficial layer(s) either outgrow from the basal cells in clumps or remain attached in scattered groups from the time of seeding on top of the basal cells (20). Three days after plating, the superficial cells usually stain positive for trypan blue, and most are removed after gentle washing of the apical surface of the culture. The superficial cells do not affect the electrical or flux measurements (13, 19, 20). In the present study, we used cultures obtained by plating at a high density to control better the timing of treatments (see below); the results were similar in cultures plated at low vs. high density (not shown).

CaSki cells were grown and maintained routinely in regular medium enriched with FCS as described previously (17, 20). In some experiments, cells were shifted to a medium enriched with FCS that was delipidized [retinoid-free medium (RFM)] to remove vitamin A. Serum delipidization was done as described previously (2, 14); briefly, FCS was added to a cooled (4°C) mixture of ethanol and acetone (1:5:5, vol/vol/vol) and mixed slowly for 4 h at 4°C. The solution was brought to room temperature and filtered twice. The protein residue was washed with ether and dried, resuspended in 2:1 vol/vol of water, frozen, and lyophilized twice for 48 h. The residue was resuspended in water to yield a protein concentration similar to the untreated serum, and aliquots of 10× PBS (pH 7.4) were added to adjust salts to isotonic conditions.

CaSki cells on filters change their permeability characteristics within the first week after plating (17). The present studies were timed for electrophysiological experiments to be conducted 4-5 days after plating (usually 4), so that it was possible to compare between different treatment groups. Because incubations in RFM required more than 6 days to exert significant effects (Ref. 14 and see RESULTS below), cells were shifted to RFM before they were plated on filters; this involved in some cases subculturing cells on regular culture dishes in RFM. For experiments, we also plated parallel filters with cells that were grown in regular culture medium to serve as controls. For each time point, we had at least three filters of test cells and three filters of parallel controls. Cells treated with retinoids were maintained in darkness until the Ussing chamber experiments.

Electrophysiological Experiments

Changes in paracellular resistance were determined in terms of changes in the transepithelial electrical resistance (RTE). For electrophysiological experiments, cells were plated on collagen-coated Anocell filters (ceramic base) as described (19), and levels of RTE were determined 4-5 days after plating on filters. Before experiments, filters containing cells were washed three times and preincubated for 10 min at 37°C in a modified Ringer buffer composed of (in mM) 120 NaCl, 5 KCl, 10 NaHCO3 (before saturating with 95% O2-5% CO2), 1.2 CaCl2, 1 MgSO4, 5 glucose, 10 HEPES, pH 7.4, and 0.1% BSA in volumes of 4.7-5.2 ml in the luminal and subluminal compartments. Drugs were added from 1,000× stocks to both the luminal and subluminal solutions; vehicles had no effect. Changes in RTE were calculated continuously across filters mounted in a modified Ussing chamber from successive measurements of the short-circuit current (Isc; normalized to the 0.6-cm2 surface area of the filter) and the transepithelial potential difference (PD; lumen negative), switching between Isc and PD at a rate of 20 Hz (19): RTE = Delta PD/Delta Isc. The experimental design of the electrophysiological measurements, including calibrations and controls, the significance of the potential and Isc, and the conditions for optimal determinations of RTE across the low-resistance CaSki epithelium were described and discussed previously (13, 19). In CaSki epithelia, the electrical resistance of the filter itself as measured in the Ussing chamber is ~20 Omega  · cm2, which is ~1.5- to 2-fold higher than the resistance generated by the cells. To determine the epithelium-generated resistance, the following steps were taken. First, in every experiment we included additional controls, such as blank filters without cells and filters containing cells grown in regular medium with known RTE based on previous studies. Second, each experiment began by adjusting the PD across a blank filter without cells to zero, to allow for subtraction of the filter-generated resistance. Third, at the conclusion of some experiments, calcium in the medium was lowered to zero by adding 2 mM EGTA to disrupt intercellular connections, including the tight junctions (19), and to abolish the paracellular resistance. Under these conditions, the RTE is similar to the resistance of the filter itself.

Determinations of the Dilution Potential

Determinations of the dilution potential (Vdil) were described previously (19). Transepithelial Vdil were determined by measuring the effect of lowering NaCl in the luminal solution on changes in voltage generated across the epithelium. This was done by replacing the Ringer buffer in the luminal compartment (130 mM NaCl) with low (10 mM) NaCl solution. Vdil is the measured PD (subluminal voltage - luminal voltage) after NaCl is lowered in the luminal solution, corrected for the potential-electrodes asymmetry. The differences in the potentials across the tips of the subluminal and the luminal electrodes were determined by the Henderson diffusion equation for monocations and monoanions after correcting for fluid resistivity (26). Changes in fluid resistivity were determined with a conductance bridge (model 31, SN 7141, Yellow Springs Instrument, Yellow Springs, OH) operating at 50-60 Hz. The Henderson diffusion equation was also used to interpret the transepithelial Vdil in terms of ionic permeabilities, i.e., the ratio uCl/uNa, where uCl and uNa are the mobilities of sodium and chloride, respectively, in the intercellular space (19).

Statistical Analysis of the Data

Data are presented as means ± SD, and significance of differences among means was estimated by Student's t-test. Trends were calculated using GB-STAT V5.3 (Dynamic Microsystems, 1995, Silver Spring, MD) and analyzed with ANOVA. Best fit of regression equations (least squares criterion) was achieved with SlideWrite Plus 1993 (Advanced Graphics Software, Carlsbad, CA), which uses the Levenberg-Marquardt algorithm, and analyzed using ANOVA. Variance among experiments (interassay variability) ranged from 15 to 27%. Variance within experiments (intra-assay variability) ranged from 5 to 8%, and all trends were consistent among experiments. Comparisons among experiments were normalized to control measurements.

Chemicals and Supplies

Anocell (Anocell-10) filters were obtained from Anotec (Oxon, UK). 9-cis-Retinoic acid, TTNPB, SRI-11217, and SRI-11237 were synthesized in the Department of Chemistry at Allergen (Irvine, CA). TTNPB is a potent synthetic RAR-selective retinoid, whereas SRI-11217 and SRI-11237 are RXR-selective synthetic retinoids (2). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Agonists were prepared as 1,000× stocks in saline or in DMSO and kept in dark conditions.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of RFM on Phase I and Phase II Responses

In CaSki cells on filters, extracellular ATP (50 µM) stimulates a biphasic change in RTE. The changes in resistance are the composite of two distinct effects (17-19): an acute decrease in the RLIS (phase I response) from 10 to 8 Omega  · cm2, followed by a slower increase in the RTJ (phase II response) to 16 Omega  · cm2 (Fig. 1A and Table 1).


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Fig. 1.   Effects of retinoid-free medium (RFM) on changes in transepithelial electrical resistance (RTE) across cultures of human cervical epithelial (CaSki) cells in response to ATP. When indicated, ATP was added at a concentration of 50 µM to the luminal and subluminal solutions from 1,000× concentrated stock (pH 7.2). A: actual tracings of changes in RTE. Early decrease in RTE is the phase I response, and late increase in RTE is the phase II response. Cells were grown in RFM for 1-11 days as described in RESULTS; cells grown in regular medium were the control (C). B: summary of the effects of RFM on ATP-induced changes in RTE. Changes in phase I response were determined relative to the trough of the decrease in RTE, and changes in phase II response were determined relative to the plateau of the increase in RTE. Changes in resistance were normalized to changes in RTE in cells grown in parallel filters in regular medium. Data are means ± SD of n = 4-5 experiments. Differences between changes in phase I and in phase II responses were significant (P < 0.01).

                              
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Table 1.   Effects of retinoid-free medium and retinoic acid on changes in transepithelial electrical resistance across cultures of human cervical cells induced by ATP, ionomycin, histamine, membrane depolarization (30 mM KCl), and diC8

To study how retinoids modulate the effects of extracellular ATP on changes in RTE, cells were grown in RFM. The timing of treatment with RFM depended on the desired length of exposure to retinoid-free conditions. For exposure of <4 days, cells plated on filters in regular culture medium were shifted to RFM for the required lengths of time. For longer exposures to retinoid-free conditions, cells grown on culture dishes were shifted to RFM and then were harvested and plated on filters in RFM for the total required length of time. For each time point, there were parallel control filters of cells that were grown in regular culture medium, and the results were normalized to these controls.

In the experiment shown in Fig. 1, cells were tested 1-11 days after treatment with RFM. For example, cells in the first time point (1 day after shifting to RFM) were plated on filters and maintained in regular culture medium for 3 days and then were shifted to RFM for 1 day before the Ussing chamber experiments. In contrast, cells at the 11-day treatment point were grown on culture dishes and shifted to RFM for 5 days; when subconfluence was reached, cells were subcultured on culture dishes in RFM for 2 additional days, harvested, and plated on filters in RFM for 4 days before the Ussing chamber experiments. Cells were therefore exposed to retinoid-free conditions for a total of 11 days. The controls were cells grown in regular culture medium in culture dishes that were then subcultured and plated on filters for the same time periods.

Incubation of cells for 11 days in RFM increased baseline RTE by 50% compared with parallel control cells that were grown in regular culture medium (Fig. 1A, Table 1) (14). To determine the effect of RFM on the ATP-induced changes in RTE, we compared the changes in phase I and phase II responses in cells grown in RFM to those obtained in cells grown in regular medium. Phase I response was determined as the acute decrease in resistance (baseline RTE minus trough of decrease in RTE); phase II response was determined as the late increase in resistance (the plateau of the increase in RTE minus baseline RTE) (Fig. 1A). Effects of retinoic acid on phase I and phase II responses were then normalized to the changes in RTE in cells grown in parallel filters in regular culture medium.

In cells grown in RFM for 11 days, both phase I and phase II responses were abolished (Fig. 1A). The loss of phase I and phase II responses was time related to the length of incubation in RFM. Phase I decreases in RTE were attenuated after 4 days of incubation in RFM and were abolished after 9-11 days. In contrast, phase II increases in RTE were attenuated already after 1 day of incubation in RFM and were abolished after 4-5 days (Fig. 1B, Table 1).

To determine whether the effects of RFM on phase I and on phase II responses are reversible, cells grown for 9 days in RFM were shifted back to regular medium for 2 days. A total of 11 days was chosen because it was the length of incubation in RFM necessary to abolish the responses to ATP (Fig. 1B). Both baseline resistance as well as the ATP-induced changes in RTE were similar to those for cells grown continuously in regular medium (Table 1).

Effects of Retinoids on Phase I and Phase II Responses.

To determine whether the effects of RFM on phase I and on phase II responses can be reversed with retinoids, cells grown in culture dishes were shifted to RFM for 5 days and then plated on filters in the same medium. After 3 days, cells were treated with 100 nM of retinoic acid for 6-48 h before the Ussing chamber experiments (total of 5 days on filters); controls were cells plated on filters in regular culture medium for 5 days. Retinoic acid had no acute effect on baseline RTE or on changes in resistance in response to ATP in cells grown in RFM (not shown), and longer incubations with the drug were needed to exert effects on resistance. Forty-eight hours after 100 nM retinoic acid was added, baseline resistance decreased from 16 to 9 Omega  · cm2 (P < 0.02) and was similar to the resistance obtained in cells grown in regular medium (P > 0.4) (14). Six to twelve hours after retinoic acid was added, phase I response was restored to near-maximal levels (Fig. 2, Table 1).


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Fig. 2.   Effects of retinoic acid on ATP-induced changes in RTE across cultures of CaSki cells. Cells grown in culture dishes were shifted to RFM for 5 days, harvested, and plated on filters in RFM for 3 days. Retinoic acid (100 nM) or vehicle (V) were added 6-48 h before the experiment as described in RESULTS. Changes in RTE (means ± SD, n = 3-6 experiments) were normalized to phase I and phase II responses obtained in cells grown in parallel filters in regular medium. Differences between changes in phase I and phase II responses were significant (P < 0.01).

In cells incubated with RFM, retinoic acid also modulated the phase II increase in resistance. In contrast to the effect of retinoic acid on phase I response, the effect of retinoic acid on phase II response began only 12 h after the drug was added, and the amplitude of phase II response was restored only to about one-half the amplitude in cells grown in regular medium (Fig. 2, Table 1).

The effects of retinoic acid on phase I and on phase II responses were dose dependent (Fig. 3). These experiments were conducted as in Fig. 1B: cells grown in culture dishes were shifted to RFM for 5 days and then plated on filters in the same medium. After 2 days, cells were treated with one of the different concentrations of retinoic acid (Fig. 3) for 2 additional days. Phase I decreases in RTE began with 10-12 M and were maximal with 10-9 M retinoic acid (Fig. 3). The dose-response curve was sigmoidal, and the data of Fig. 3 were fitted to a modified Hill equation with half-maximal effective concentration (K1/2) of retinoic acid of 0.12 ± 0.03 nM and a Hill coefficient (n) of 1.1 ± 0.3 (P < 0.01). Phase II increases in RTE began at retinoic acid concentrations of 10-10 nM and reached saturation at 10-8 nM (Fig. 3). The amplitude of the restored phase II response was about one-half of the amplitude of phase II response in cells grown in regular medium (Fig. 3). The dose-response curve for phase II response was sigmoidal, and the data of Fig. 3 were also fitted to a modified Hill equation with K1/2 of retinoic acid of 1.0 ± 0.2 nM and n of 1.1 ± 0.2 (P < 0.01). These results indicate that retinoic acid has a more potent and efficacious effect on the phase I than on the phase II response and that phase II response in cells incubated in RFM can be restored only in part by retinoic acid.


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Fig. 3.   Dose-response relationship between retinoic acid and changes in RTE in response to ATP in CaSki cultures. Cells were grown for 7 days in RFM and treated for 2 additional days with retinoic acid at concentrations ranging from 10-12 to 10-6 M as described in RESULTS. Changes in RTE were normalized to phase I and phase II responses obtained in cells grown for 9 days in regular medium. There were a total of 6 experiments; data of individual experiments were fitted to a modified Hill equation: RTE = RTE(max) · 1/{1 + (K1/2/[RA])n} + RTE(min) · (1 - 1/{1 + (K1/2/[RA])n}) to determine 4 parameters: RTE(max) (the maximal resistance), RTE(min) (the minimal resistance), K1/2 {retinoic acid concentration ([RA]) that produces half-maximal effect}, and n (the Hill coefficient, which is related to the number of retinoid binding sites). For each of the phase I and phase II responses, experimental data are means ± SD and the theoretical curve is given by the means of the 4 measured parameters. Differences between changes in phase I and in phase II responses were significant (P < 0.01).

In addition to retinoic acid, other retinoids also modulated the effects of ATP on RTE. These experiments were conducted in a similar manner, as was described above for Fig. 3. TTNPB (an RAR-selective ligand), SRI-11217 and SRI-11237 (RXR-selective ligands), and 9-cis-retinoic acid (a panagonist; i.e., binds both RAR and RXR subtypes) restored phase I response in a dose-related manner, similar to the effect of retinoic acid (Fig. 4A). For each of the retinoids, the K1/2 was ~0.2 nM and n was ~1.0. In contrast to the effect on the phase I response, only 9-cis-retinoic acid and TTNPB restored the late increase in RTE in response to ATP (Fig. 4A), with K1/2 of 2 nM (9-cis retinoic acid) and 3 nM (TTNPB) and n of 1.2 and 1.1, respectively.


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Fig. 4.   Effects of different retinoids on changes in RTE across cultures of CaSki cells in response to ATP. A: dose-response relationship between different retinoids and changes in phase I (solid symbols) and phase II (open symbols) responses. C, control (vehicle); 9-cis RA, 2-cis-retinoic acid. Experiments were done as in Fig. 3, and changes in RTE were normalized to phase I and phase II responses obtained in cells grown for 9 days in regular medium. B: means ± SD (n = 3-5) of the effects of different retinoids, all added at 100 nM, on changes in phase I (solid bars) and phase II (open bars) responses. RA, retinoic acid. Phase I decreases in RTE in cells treated with any of the retinoids were similar (P > 0.2) and significantly greater than in cultures treated with the vehicle only (P < 0.01). Phase II increases in RTE in cells treated with retinoic acid, 9-cis-retinoic acid, or TTNPB were similar (P > 0.4) and significantly greater than in cultures treated with the vehicle or with SRI-11217 or SRI-11237 (P < 0.01).

To determine the efficacy of the RAR- vs. RXR-selective ligands in restoring changes in resistance in response to ATP, cells grown in RFM were treated with retinoids, added at a concentration of 100 nM, which produces maximal effects on phase I or phase II response (Figs. 3 and 4A). Each of the tested retinoids restored the phase I decrease in RTE to near the same degree as in cells grown in a regular medium (Fig. 4B). Only retinoic acid, 9-cis-retinoic acid, and TTNPB restored phase II increases in RTE, and the amplitude of the restored phase II response was about one-half of that in cells grown in regular medium (Fig. 4B).

Mechanisms of Retinoid Effects

Effects of actinomycin D on ATP-induced changes in RTE. Retinoids modulate cell functions via several known mechanisms, including transcription regulation (2). The objective of the next experiment was to study the effects of the transcription inhibitor actinomycin D on the ATP-induced phase I and phase II responses in cells treated with retinoic acid. Cells grown in culture dishes were shifted to RFM for 5 days, then plated on filters in the same medium for 2 days, and treated with 100 nM retinoic acid for 2 additional days. Actinomycin D (10 µM) was added for 0.5-2.5 h before the Ussing chamber experiments. The controls were cells grown in regular culture medium in culture dishes and filters for the same time periods that were treated with actinomycin D or with the vehicle 0.5-2.5 h before the Ussing chamber experiments.

For day 4 control cells on filters, actinomycin D had no effect on baseline resistance and on the phase I response to ATP. In contrast, incubation with actinomycin D for 2.5 h attenuated the phase II response by ~25% compared with cells not treated with actinomycin D: a net increase in RTE in response to ATP of 4.5 ± 0.5 vs. 6.2 ± 0.5 Omega  · cm2 (P < 0.03), respectively.

For day 4 cells that were previously grown in RFM for 7 days and treated for 2 additional days with retinoic acid, actinomycin D had no effect on baseline resistance (not shown) and on the retinoic acid-restored phase I response (Fig. 5). In contrast, actinomycin D abrogated the retinoic acid-restored phase II response. Incubation with actinomycin D for 2.5 h resulted in a net increase in RTE in response to ATP of only 1.1 ± 0.3 Omega  · cm2, which is only 20% of the increase in resistance in control cells treated with actinomycin D (Fig. 5).


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Fig. 5.   Effects of actinomycin D on changes in RTE in response to ATP across retinoid-treated CaSki cells. Cells were grown for 7 days in RFM and treated for 2 additional days with 100 nM retinoic acid as described in RESULTS. Actinomycin D (10 µM) was added to both the luminal and subluminal solutions for the designated times before ATP was added. Data of changes in RTE (means ± SD, n = 3) were normalized to phase I and phase II responses obtained in day 4 cells grown in regular medium and treated with actinomycin D or vehicle. Differences between changes in phase I and in phase II responses were significant (P < 0.01).

Effects of ionomycin, histamine, KCl, and 1,2-dioctanoyl-sn-diglycerol on changes in RTE. The objective of the next set of experiments was to determine how retinoids modulate the effects of agents that produce phase I-like and phase II-like responses by bypassing the P2U receptor(s). Phase I-like decreases in RTE were induced by ionomycin and by histamine; phase II-like increases in RTE were induced by KCl-dependent membrane depolarization (18) or by 1,2-dioctanoyl-sn-diglycerol (diC8) (17). Cells grown in culture dishes were shifted to RFM for 5 days, then plated on filters in the same medium for 2 days, and treated with 100 nM retinoic acid or the vehicle for 2 additional days. The controls were cells grown in regular culture medium in culture dish and filters for the same time periods.

Ionomycin decreased RTE by ~5 Omega  · cm2, and the effect was similar in cells grown in RFM and treated with retinoic acid or with the vehicle and in cells grown in regular medium (P > 0.3, Fig. 6A, Table 1). Histamine decreased RTE transiently by ~2.5 Omega  · cm2, and the effect was similar in cells grown in RFM and treated with retinoic acid or with the vehicle and in cells grown in regular medium (P > 0.3, Fig. 6B, Table 1).


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Fig. 6.   Effects of ionomycin (A) and histamine (B) on RTE across cultures of CaSki cells. Cells were grown for 7 days in RFM and treated for 2 additional days with 100 nM retinoic acid (RFM/RA) or the vehicle (RFM) as described in RESULTS. Cells grown in regular medium served as control (C). Agonists [ionomycin (5 µM) and histamine (100 µM)] were added to both the luminal and subluminal solutions from 1,000× stocks. Experiments were repeated 3 times.

Increasing extracellular KCl to 30 mM induced a biphasic change in RTE, similar to the effect of ATP (Fig. 7A). KCl decreased RTE by ~3 Omega  · cm2 in cells grown in RFM and treated with retinoic acid or with the vehicle and in cells grown in regular medium (P > 0.3, Fig. 7A, Table 1). The KCl-induced late increase in RTE differed among the three groups of cells: in control cells, RTE increased by 7.6 Omega  · cm2 (P < 0.01, Fig. 6B, Table 1); in cells grown in RFM, and regardless of treatment with retinoic acid, RTE increased only by 4 Omega  · cm2 (P > 0.01, Fig. 7A, Table 1).


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Fig. 7.   Effects of KCl (A) and 1,2-dioctanoyl-sn-diglycerol (diC8; B) on RTE across cultures of CaSki cells. Experiments were done as in Fig. 6. Agonists [KCl (added to increase medium level from 5 to 30 mM) or diC8 (10 µM)] were added to both the luminal and subluminal solutions from 1,000× stocks. Experiments were repeated 3-4 times.

diC8 increased RTE, but the effect differed among the three groups of cells: in control cells, diC8 increased RTE by 7.7 Omega · cm2 (P < 0.01, Fig. 7B, Table 1); in cells grown in RFM, and regardless of treatment with retinoic acid, diC8 increased RTE by only 4 Omega  · cm2 (P > 0.02, Fig. 7B, Table 1). These results indicate that exposure to retinoid-free conditions attenuates the increases in resistance induced by membrane depolarization or by diC8.

In cells grown in RFM, and regardless of treatment with retinoic acid, diC8 had no additional effect on RTE when added during the plateau of the ATP-induced phase II increase in resistance (not shown), indicating that the effects of ATP (phase II increase in resistance) and diC8 are nonadditive.

To determine whether actinomycin D affects the changes in RTE in response to KCl or diC8, cells were treated with 10 µM actinomycin D for 1 h before the Ussing chamber experiments. Actinomycin D did not change baseline resistance significantly, and neither KCl nor diC8 induced changes in resistance in any of the three groups of cells (not shown).

Modulation of RLIS and RTJ. To determine whether in retinoid-treated cells ATP activates changes in RLIS or in RTJ, we measured the effects of ATP on changes in the Vdil and we calculated the effects on the ratio of mobilities of chloride and sodium (uCl/uNa). The rationale was that cation selectivity is a property of the tight junctions, and changes in uCl/uNa reflect modulation of RTJ (24). Cells grown in culture dishes were shifted to RFM for 5 days, then plated on filters in the same medium for 2 days, and treated with 100 nM retinoic acid or the vehicle for 2 additional days. The controls were cells grown in regular culture medium in culture dishes and filters for the same time periods. Before ATP was added, NaCl in the luminal side was lowered from 130 to 10 mM to establish a Vdil, and changes in uCl/uNa were determined as described in METHODS. The level of uCl/uNa across cells incubated in RFM plus retinoic acid was 1.34 ± 0.09, similar to those across control cells (1.33 ± 0.08, P > 0.4, Fig. 8). In cells exposed to low NaCl in the luminal solution, ATP produced biphasic changes in RTE similar to those obtained under symmetrical NaCl concentrations of 130 mM (compare Figs. 8 and 1A). The uCl/uNa decreased in a time-related manner after ATP was added, and the decrease in uCl/uNa correlated in time with the phase II response but not with the phase I response (Fig. 8), indicating that phase II increases in RTE in retinoid-treated cells are associated with increased cation selectivity.


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Fig. 8.   Effects of retinoic acid on the ATP-induced changes in RTE and in the mobility ratio of chloride to sodium (uCl/uNa). Experiments were done as in Fig. 6. Before ATP was added, solution in the luminal side was replaced with low-NaCl buffer, thus lowering NaCl in the luminal solution from 130 to 10 mM. Changes in uCl/uNa were determined from the established dilution potential, and levels of RTE were determined as described in METHODS. Experiment was repeated 3 times.

In cells grown in RFM plus retinoic acid, the ratio of uCl to uNa decreased from 1.34 ± 0.09 to 1.31 ± 0.09 [Fig. 8, P < 0.01 (paired t-test)]. In cells grown in regular medium, the ratio of uCl to uNa decreased from 1.33 ± 0.08 to 1.26 ± 0.09 (Fig. 8, P < 0.01). The differences between the decreases of uCl/uNa across cells grown in RFM plus retinoic acid and those across control cells were significant (P < 0.01).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present results indicate that retinoids modulate the effects of extracellular ATP on paracellular resistance in human cervical cells. The experiments were done with human CaSki cells, which are a good model for the endocervical epithelium, based on morphological, biochemical, and molecular criteria (20). Also, the permeability characteristics of the cultured cervical cells are similar to those of the endocervical epithelium (14).

The present results indicate that RFM (i.e., vitamin A deficiency) abrogates and retinoids restore the ATP-induced phase I decrease in RLIS and the phase II increase in RTJ. Neither vitamin A deficiency nor retinoids had toxic effects on the cells because 1) phase I- and phase II-like changes in RTE could be evoked in vitamin A-deficient cells with agents that act distal to the P2U receptor(s), regardless of treatment with retinoids, and 2) shifting cells grown in RFM to a medium enriched with retinoid-containing serum restored baseline resistance and the changes in RTE in response to ATP. The data further support the theory that phase I and phase II changes in RTE are mediated by two distinct signal mechanisms (17, 18) because phase I and phase II responses differed with regard to 1) the time course of the effects of vitamin A deficiency and retinoid treatment on changes in RTE, 2) the retinoid agonists profile, 3) the dose requirements for retinoids necessary to restore phase I and phase II responses, and 4) the responses to actinomycin D.

Incubation of cervical cells in medium with delipidized serum resulted in an increased baseline RTE, and it abrogated phase I response; however, it had no effect on phase I-like decreases in resistance induced by ionomycin and histamine. It was previously suggested that decreases in RTE induced by ATP, ionomycin, and histamine are mediated by a common signaling mechanism (17, 18), and the present results suggest that retinoids modulate a proximal step in the ATP-dependent stimulus, such as the P2U-purinergic receptor.

Incubation of cervical cells in medium with delipidized serum also abrogated phase II response and attenuated the increases in RTJ in response to KCl or diC8. Neither of these effects could be reversed completely by retinoic acid. This is in contrast to the effect of retinoic acid on baseline resistance (14). It was previously shown (14), and confirmed in the present study, that delipidized serum increases baseline paracellular resistance mainly by increasing RTJ. In addition, retinoic acid reversed the effect of delipidized serum and decreased RTJ (14). These results suggest that delipidized serum and retinoids have a number of different effects on the tight junctions in human cervical cells and they may modulate RTJ by different mechanisms, e.g., regulation of the junctional complexes (which determine the baseline resistance), vs. regulation of the P2U receptor signaling cascade that activates an increase in RTJ (phase II response).

The molecular mechanisms of retinoid action on cervical cells are only partially understood. Retinoids act via nuclear receptors that are known to be expressed in human cervical cells (1-3, 8, 11, 21). The present results suggest that the phase II increase in resistance is mediated via nuclear retinoid receptors. 1) Retinoic acid restored the phase II response within 6-12 h of treatment, a length of time compatible with transcription regulation. 2) The low concentration of retinoic acid (K1/2 of 1 µM) correlates with the dissociation constant of the retinoid receptors (9, 10). 3) The dose-response curve of increase in resistance in response to ATP vs. retinoic acid concentrations was saturable and was fitted by a modified Hill equation with n = 1. 4) Phase II response was restored in cells treated with RAR-selective or RAR/RXR-selective ligands but not by RXR-selective ligands. 5) Actinomycin D inhibited the retinoic acid-induced effect on phase II response but not the KCl- or diC8-induced increases in resistance. It was suggested that KCl induces an increase in RTJ by stimulating calcium influx, an early step in the phase II response (18). A possible interpretation of the present data is therefore that retinoids, acting via an RAR, upregulate transcription or inhibit degradation of a message in the signaling cascade proximal to calcium influx.

In contrast to the effect on phase II response, longer incubation in RFM was required to eliminate phase I response (4 vs. 11 days, respectively), and phase I response was restored earlier after retinoic acid was added (<6 h). Also, retinoic acid had a more potent effect on the phase I response (K1/2 of 0.1 µM). Moreover, the phase I response was restored both with RAR- and with RXR-selective retinoids, and actinomycin D had little effect on the phase I response. Interestingly, vitamin A deprivation eliminated phase I response but not the ionomycin-induced decrease in resistance. This suggests that vitamin A deficiency downregulates and that retinoids upregulate necessary step(s) in the signaling cascade proximal to calcium mobilization.

Vitamin A and related ligands produce multiple effects on cervical epithelial cells (1-3, 8, 11, 21). The present study adds to this list and suggests that retinoids determine the degree of RTJ of endocervical cells and regulate the responses to extracellular ATP. Because plasma levels of vitamin A are tightly regulated (9, 10), the net effect on transcervical permeability may depend on changes in the expression of nuclear RAR or RXR, changes in retinoid metabolism, and/or the concentration of ATP in the extracellular fluid. Previous studies in rodents suggest that levels of retinoid receptors in the cervix change during the estrous cycle (5, 28, 29). The present results therefore raise the possibility that sex steroid hormones regulate cervical mucus secretion indirectly by modulating tissue levels of retinoid receptors and that the retinoid receptors, in turn, control the degree of occlusion of the intercellular space by the tight junctions and the responses to extracellular ATP.

Vitamin A-dependent regulation of baseline paracellular resistance and the responses to ATP may be important in vivo for regulation of cervical mucus because even small changes in RTE may have profound effects on fluid and solute secretion. Release of ATP into the extracellular space from nerve endings or from cells that participate in the inflammatory process can stimulate the phase I response and decrease RLIS and therefore can enhance secretion of fluids and solutes from the blood into the cervical canal. Vitamin A would tend to increase transcervical permeability because retinoids decrease baseline RTJ, and phase I decreases in RLIS are more sensitive to the effects of retinoids than the phase II increases in RTJ (present results). These data correlate with the observations in vivo that vitamin A deficiency leads to dryness of the endocervix (i.e., diminished transudation of fluids from the blood into the lumen across the cervical epithelium), whereas treatment with vitamin A reverses the condition (9, 10).

The role in vivo of phase II increases in RTJ, on the other hand, is less well understood. An ATP-induced increase in RTJ would tend to decrease transcervical transport and diminish production of cervical mucus. It is possible that, under certain pathological conditions in vivo, e.g., inflammation of the cervix, that are associated with excessive secretion of cervical mucus (12), the phase II response will tend to diminish the secretion of fluids from blood into the lumen.

    ACKNOWLEDGEMENTS

This study was supported by National Institute of Child Health and Human Development Grants HD-00977 and HD-29924 (to G. I. Gorodeski) and a grant from the American Institute for Cancer Research (to R. L. Eckert). These studies used the facilities of the Skin Diseases Research Center of Northeast Ohio (National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-39750).

    FOOTNOTES

Present address of P. Burfeind: Institute of Human Genetics, Georg-August-University, Gosslerstr. 12D, D-37073 Goettingen, Germany

Address for reprint requests: G. I. Gorodeski, Univ. MacDonald Womens Hospital, Univ. Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106.

Received 4 November 1997; accepted in final form 9 January 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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