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

C. Deachapunya and S. M. O'Grady

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 (I_sc) 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 µM PGE₂ increased I_sc by 41 ± 3 µA. 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 I_sc, 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 I_sc 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 I_sc 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-I_sc 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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