Regulation of chloride secretion across porcine endometrial epithelial cells by prostaglandin E2.
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.
Deachapunya
C
C
Department of Physiology, University of Minnesota, St Paul, MN 55108, USA.
O'Grady
S M
SM
D000595:Amino Acid Sequence; D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D000838:Anions / Q000378:metabolism; D000906:Antibodies / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D015232:Dinoprostone / Q000276:immunology / Q000494:pharmacology*; D004717:Endometrium / Q000187:drug effects / Q000502:physiology*; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D005260:Female; D005665:Furosemide / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D009579:Nitrobenzoates / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D028021:Sodium-Potassium-Chloride Symporters; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors
D000838:Anions; D000906:Antibodies; D002352:Carrier Proteins; D018118:Chloride Channels; D002712:Chlorides; D009579:Nitrobenzoates; D010446:Peptide Fragments; D028021:Sodium-Potassium-Chloride Symporters; D013873:Thionucleotides; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D005665:Furosemide; D000666:Amphotericin B; D000242:Cyclic AMP; D015232:Dinoprostone; D005905:Glyburide