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. 1998 Apr 1;508 ( Pt 1)(Pt 1):31-47.
doi: 10.1111/j.1469-7793.1998.031br.x.

Regulation of chloride secretion across porcine endometrial epithelial cells by prostaglandin E2

C Deachapunya  1 , S M O'Grady
Affiliations

Regulation of chloride secretion across porcine endometrial epithelial cells by prostaglandin E2

C Deachapunya et al. J Physiol. .

Abstract

1. The objective of this study was to investigate the mechanism of PGE2 regulation of Cl- transport across glandular endometrial cells grown in primary culture. 2. Most of the basal short circuit current (Isc) was inhibited by luminal addition of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or glibenclamide, suggesting the presence of a basally active Cl- conductance in the apical membrane. 3. Basolateral addition of 10 microM PGE2 increased Isc by 41 +/- 3 microA. A similar response was observed when cells were treated with 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Pretreatment of monolayers with NPPB and glibenclamide blocked the PGE2 and cAMP-mediated increase in Isc, suggesting that the effects of PGE2 and cAMP were dependent on the activity of an apical NPPB- and glibenclamide-sensitive conductance. 4. Addition of 50 nM antiPGE2 antibody to the basolateral bathing solution decreased basal Isc by 20 % and shifted the threshold response to exogenous PGE2. This result suggests autocrine regulation of electrogenic Cl- transport by PGE2. 5. Experiments with amphotericin B-permeabilized monolayers revealed that the apical PGE2-activated, NPPB- and glibenclamide-sensitive conductance was Cl- dependent and that the current-voltage relationship and anion permeation properties (SCN->Br- > Cl- > I-) were characteristic of the cystic fibrosis transmembrane conductance regulator (CFTR). 6. Cultured porcine endometrial epithelial cells were specifically labelled with an antibody to a peptide sequence within the regulatory domain of CFTR. 7. The effect of PGE2 was blocked by basolateral addition of bumetanide and furosemide at concentrations that are selective for inhibition of Na+-K+-2Cl-cotransport activity. The effect of bumetanide on Isc was Cl- dependent, suggesting a role for the bumetanide-sensitive transport pathway in Cl- secretion. 8. PGE2 and cAMP also activated an outwardly rectifying basolateral K+ channel which presumably sustains the driving force for electrogenic Cl- efflux across the apical membrane. 9. The concentration-conductance and concentration-Isc response relationships for PGE2 showed that basolateral K+ permeability was rate limiting with respect to transepithelial anion secretion and that activation of a basolateral K+ channel by PGE2 was necessary to achieve maximum rates of Cl- secretion.

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Figures

Figure 1
Figure 1. Isolation and identification of glandular endometrial epithelial cells using cytokeratin antibody
A and B, phase-contrast micrographs of endometrial epithelial glands after isolation and primary culture of epithelial cells grown on plastic after 4 days. C and D, localization of cytokeratin antibody labelling using laser confocal immunofluorescence microscopy of cultured glandular endometrial epithelial cells. The cells were grown on lab-tek slides in standard media for 5 days. Monolayers were fixed, permeabilized and incubated with mouse antibody against cytokeratin peptide 18 and goat antimouse Cy3-labelled secondary antibody (C). D, cells incubated with Cy3-labelled secondary antibody alone. Scale bar, 25 μm; applies to all panels.
Figure 2
Figure 2. Time course showing changes in transepithelial resistance of the cell monolayers over a period of 20 days
After a week in culture, the cells were seeded on permeable supports. The transepithelial resistance was measured using the EVOM epithelial voltohmmeter coupled to Ag-AgCl ‘chopstick’ electrodes. The transpithelial resistance reached a maximum value of 2870 ± 54 Ω cm2 after 3 days and was sustained up to 20 days (n= 100, N= 10).
Figure 3
Figure 3. Effects of Cl− channel inhibitors on basal Isc
A, a representative Isc tracing showing the effect of NPPB on basal Isc. Addition of 100 μm NPPB to the apical solution produced a rapid and sustained decrease in basal Isc by 79 ± 5 %. The subsequent addition of 100 μm NPPB further decreased basal Isc whereas the addition of 200 μm glibenclamide (Glib) produced no further response. Addition of 3 μm PGE2 to the basolateral solution had no stimulatory effect in the presence of NPPB and glibenclamide in the apical solution (n= 9, N= 4). B, concentration-response relationships showing the decrease in Isc following treatment with various concentrations of NPPB and glibenclamide to the apical solution. The IC50 value was 19 μm for NPPB and 52 μm for glibenclamide (n= 8, N= 4 for each compound). NPPB was found to be 2.7-fold more potent than glibenclamide.
Figure 4
Figure 4. Representative Isc tracings showing the effect of PGE2 on basal Isc
Addition of 3 μm PGE2 to the basolateral solution produced an increase in Isc followed by a sustained plateau greater than that of the basal Isc. The PGE2-stimulated Isc was blocked by apical additions of 200 μm NPPB (n = 7, N = 4) (A) and 200 μm glibenclamide (Glib) (n = 5, N = 4) (B). In these experiments the cells were grown under standard media conditions for 7 days followed by Phenol Red-free medium containing charcoal-stripped serum for 7 days (see Methods).
Figure 5
Figure 5. Concentration-response relationships for PGE2 and PGF2α and effect of CPT-cAMP on basal Isc
A, concentration-response relationships showing the increase in Isc following treatment with various concentrations of PGE2 and PGF2α to the basolateral solution. The EC50 value was 72 nm for PGE2 (n= 10, N= 5) and > 7.5 μm for PGF2α (n= 11, N= 4). B, a representative Isc tracing showing the effect of CPT-cAMP on basal Isc. Addition of 100 μm CPT-cAMP to the basolateral solution produced an immediate increase in Isc followed by a sustained plateau greater than that of the basal Isc. The cAMP-stimulated Isc was blocked by apical additions of 200 μm NPPB (n= 6, N= 3).
Figure 6
Figure 6. Effects of anion substitution and antiPGE2 antibody on Isc
A, effect of anion substitution on the maximal Isc response to PGE2. In standard porcine Ringer solution, PGE2 at a concentration of 10 μm produced a mean increase in Isc of 41 ± 3 μA (n= 15, N= 3). Replacement of Cl− and HCO3− in both the apical and basolateral solutions significantly inhibited the maximal response to PGE2 by 79 % and 96 %, respectively (P < 0.001 compared with control). The PGE2 response was completely abolished following replacement of both Cl− and HCO3− (n= 5, N= 3 for each condition). B, a representative Isc tracing showed the effect of antiPGE2 antibody on basal Isc. Addition of 50 nm antiPGE2 antibody to the basolateral solution inhibited basal Isc by 21 ± 7 % (n= 5, N= 2). Addition of 10 μm indomethacin (Indo) to both the apical and the basolateral solution further inhibited the basal current. Pretreatment with antiPGE2 antibody blocked the response to exogenous PGE2 at concentrations of 3 and 30 nm.
Figure 7
Figure 7. Current-voltage (I-V) relationships for the NPPB- and glibenclamide-sensitive conductances in the apical membrane
A, a representative tracing of the glibenclamide-sensitive current obtained from amphotericin B-permeabilized monolayers in response to a voltage step protocol from -90 to +120 mV (15 mV step increments) at a holding potential of 0 mV. Glibenclamide (200 μm) was added to the apical solution. B, NPPB- and glibenclamide-sensitive components of the apical membrane current were plotted as a function of voltage. Experiments were performed under conditions where the basolateral surface was permeabilized with amphotericin B and bathed in KMeSO4 Ringer solution with 10 mm NaCl. Standard Ringer solution was used to bathe the apical surface of the epithelium. NPPB or glibenclamide, at a concentration of 200 μm, was added to the apical solution. Mean reversal potentials for NPPB- and glibenclamide-sensitive currents were -28 ± 3 and -27 ± 1 mV, respectively (n= 7, N= 4 for each experiment).
Figure 8
Figure 8. Current-voltage (I-V) relationships for the PGE2-, NPPB- and glibenclamide-sensitive conductances in the apical membrane
A, a representative tracing of the glibenclamide-sensitive component of the PGE2-stimulated current. Current measurements were obtained from amphotericin B-permeabilized monolayers which were held at 0 mV and stepped through a series of voltages from -90 to +50 mV (10 mV step increments). PGE2 (3 nm) was added to the basolateral surface and NPPB or glibenclamide, at a concentration of 200 μm, was subsequently added to the apical solution. B, PGE2-sensitive, NPPB-sensitive and glibenclamide-sensitive currents were plotted as a function of voltage. Experiments were performed using amphotericin B-permeabilized monolayers (basolateral surface) with KMeSO4 Ringer solution containing 10 mm NaCl bathing the basolateral surface and standard Ringer solution bathing the apical surface. Mean reversal potentials for PGE2-sensitive, NPPB-sensitive and glibenclamide-sensitive currents were -28 ± 1 mV (n= 13, N = 4), -30 ± 2 mV (n= 6, N = 3) and -31 ± 2 mV (n= 7, N = 4), respectively.
Figure 9
Figure 9. The reversal potential for PGE2-sensitive, NPPB-sensitive and glibenclamide-sensitive currents at various basolateral Cl− concentrations
The data were fitted using linear regression analysis with correlation coefficients (R) of 0.991, 0.993 and 0.999 for PGE2-, NPPB- and glibenclamide-sensitive responses, respectively (n= at least 5, N= 4 for each experiment).
Figure 10
Figure 10. Current-voltage (I-V) relationships for the CPT-cAMP, NPPB- and glibenclamide-sensitive conductances in the apical membrane
A, a representative tracing of glibenclamide-sensitive current after CPT-cAMP stimulation. The current was obtained from amphotericin B-permeabilized monolayers which were held at 0 mV and stepped through a series of voltages from -100 to +70 mV (10 mV increments). CPT-cAMP (1 μm) was added to the basolateral solution and NPPB or glibenclamide at a concentration of 200 μm was subsequently added to the apical solution. B, cAMP-sensitive, NPPB-sensitive and glibenclamide-sensitive currents were plotted as a function of voltage. Experiments were performed using amphotericin B-permeabilized monolayers (basolateral surface) with KMeSO4 Ringer solution containing 10 mm NaCl bathing the basolateral surface and standard Ringer solution bathing the apical surface of the epithelium. Average reversal potentials for CPT-cAMP activation, NPPB inhibition and glibenclamide inhibition were -29 ± 1 mV (n= 10, N= 3), -38 ± 2 mV (n= 5, N= 3) and -30 ± 1 mV (n= 5, N= 3), respectively.
Figure 11
Figure 11. Current-voltage (I-V) relationships of PGE2-sensitive current in the presence of 50 mm KCl in the intracellular (basolateral) solution and 50 mm KCl, KSCN, KBr or KI in the extracellular (apical) solution
Experiments were performed using amphotericin B to permeabilize the basolateral membrane. PGE2 was added to the basolateral solution at 3 μm for KSCN and KI experiments and at 3 nm for KBr and KCl experiments. Average reversal potentials for KSCN, KBr, KCl and KI were -23 ± 2 mV (n= 5, N= 3), -5 ± 0.5 mV (n= 6, N= 3), 0 ± 0.2 mV (n= 6, N= 3) and 18 ± 1 mV (n= 5, N= 3), respectively.
Figure 12
Figure 12. Localization of CFTR-like immunoreactivity using laser confocal immunofluorescence microscopy of cultured glandular endometrial epithelial cells
The results shown are the summation of images (4–6) starting from the permeable support towards the apical membrane in 3 μm increments. The cells were grown on Lab-tek slides (A, C and E; scale bar, 25 μm) or permeable filter supports (B, D and F; scale bar, 12.5 μm) in standard media (A and B) and Phenol Red-free medium containing charcoal-stripped serum (B and F). Monolayers were fixed, permeabilized and labelled with rabbit antibody against a peptide sequence within the R-domain of human CFTR and goat antirabbit Cy3-labelled secondary antibody (A and D. E) and F show the results when CFTR antibody was pre-incubated with CFTR peptide antigen.
Figure 13
Figure 13. Current-voltage (I-V) relationships of the PGE2-sensitive permeability in the basolateral membrane
A, a representative tracing of PGE2-sensitive current obtained from amphotericin B-permeabilized monolayers in response to step commands from -100 to +70 mV (10 mV increments) at a holding potential of 0 mV. PGE2 (3 μm) was added to the basolateral solution. B, effects of PGE2 concentrations on PGE2-sensitive currents obtained from amphotericin B-permeabilized monolayers in response to step commands from -100 to +70 mV in 10 mV increments at a holding potential of 0 mV. Experiments were performed using amphotericin B-permeabilized monolayers (apical surface) with KMeSO4 Ringer solution containing 10 mm NaCl bathing the apical surface and standard Ringer solution bathing the basolateral surface. The mean reversal potential was -59 ± 2 mV (n= 5, N= 3 for each experiment).
Figure 14
Figure 14. Current-voltage (I-V) and concentration-response relationships for the cAMP-sensitive conductance in the basolateral membrane
A, effects of increasing CPT-cAMP concentrations on the basolateral outwardly rectifying K+ conductance. The mean reversal potential was -61 ± 4 mV (n= 5, N= 3). B, concentration-response relationship showing the increase in basolateral membrane conductance following treatment with increasing concentrations of CPT-cAMP added to the basolateral solution. The EC50 value was 21 μm (n= 5, N= 3).
Figure 15
Figure 15. Concentration-Isc and concentration-conductance response relationships following addition of increasing concentrations of PGE2 to the basolateral solution
The EC50 values were 14 nm for the apical conductance (n= 6, N= 3), 73 nm for Isc (n= 10, N= 5) and 81 nm for the basolateral membrane conductance (n= 5, N= 3).
Figure 16
Figure 16. Effects of bumetanide and furosemide on basal Isc
A, a representative Isc tracing showing the effect of bumetanide on basal Isc. Addition of 100 μm bumetanide to the basolateral solution produced a sustained decrease of 41 ± 5 % in basal Isc (n= 7, N= 4). B, concentration-response relationships showing the decrease in Isc following treatment with various concentrations of bumetanide and furosemide to the basolateral solution. The IC50 values were 1.5 μm for bumetanide and 75 μm for furosemide (n= 5, N= 3 for each compound). Bumetanide was found to be at least 50-fold more potent than furosemide.
Figure 17
Figure 17. Histogram illustrating the percentage decrease of bumetanide and furosemide on basal and PGE2-stimulated Isc in standard porcine Ringer solution
Addition of 100 μm bumetanide to the basolateral solution decreased the basal Isc by 41 ± 5 % (n= 7, N= 4) and inhibited 88 ± 13 % of PGE2-stimulated Isc response (n= 5, N= 3, P < 0.001). Basolateral addition of 100 μm furosemide decreased the basal Isc by 37 ± 2 % (n= 4, N= 4) and inhibited 83 ± 11 % of PGE2-stimulated Isc response (n= 8, N= 3, P < 0.001).
Figure 18
Figure 18
Proposed model explaining the mechanism of action of PGE2 on Cl− secretion across endometrial epithelial cells (see Discussion). AC, adenylyl cyclase; G, G protein.
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