title: Forskolin-induced cell shrinkage and apical translocation of functional enhanced green fluorescent protein-human alphaENaC in H441 lung epithelial cell monolayers.
abstract: Elevation of intracellular cAMP increases fluid re-absorption in the lung by raising amiloride-sensitive Na+ transport through the apically localized epithelial, amiloride-sensitive Na+ channel (ENaC). However, the signaling pathways mediating this response are still not fully understood. We show that inhibition of protein-tyrosine kinase (PTK) with Genistein and protein kinase A (PKA) with KT5720, decreased forskolin-stimulated amiloride-sensitive short circuit current (I(sc)) across H441 adult human lung epithelial cell monolayers. KT5720 also decreased basal I(sc). Stable expression of green fluorescent protein (GFP)-labeled human alphaENaC in H441 cells was used to investigate dynamic changes in the cellular localization of this protein in response to forskolin. Reverse transcription-PCR and immunoblotting analysis revealed two clones expressing a truncated (alphaC3-5) and full-length (alphaC3-3) EGFP-halphaENaC protein. Only the alphaC3-3 clone displayed dome formation and exhibited a 50% increase in basal and forskolin-stimulated amiloride-sensitive I(sc) indicating that the full-length protein was required for functional activity. Apical surface biotinylation and real-time confocal microscopy demonstrated that EGFP-halphaENaC (alphaC3-3) translocated to the apical membrane in response to forskolin in a Brefeldin A-sensitive manner. This effect was completely inhibited by Genistein but only partially inhibited by KT5720. Forskolin also induced a reduction in the height of cells within alphaC3-3 monolayers, indicative of cell shrinkage. This effect was inhibited by KT5720 but not by Genistein or Brefeldin A. These data show that forskolin activates PKA-sensitive cell shrinkage in adult human H441 lung epithelial cell monolayers, which induces a PTK-sensitive translocation of EGFP-halphaENaC subunits to the apical membrane and increases amiloride-sensitive Na+ transport.
authors.affiliation: Division of Basic Medical Sciences, Ion Channels and Cell Signaling Centre, St. Georges' University of London, Cranmer Terrace, Tooting, London SW17 0RE, United Kingdom.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Woollhead
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Baines
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D001692:Biological Transport; D019921:Biotinylation; D020126:Brefeldin A / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D053503:Epithelial Sodium Channels; D019833:Genistein / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D007477:Ions; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D011758:Pyrroles / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000737:chemistry; D015222:Sodium Channels / Q000378:metabolism*; D014162:Transfection
chemical_list: D002227:Carbazoles; D053503:Epithelial Sodium Channels; D007211:Indoles; D007477:Ions; D011758:Pyrroles; D012333:RNA, Messenger; D015222:Sodium Channels; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005576:Colforsin; D020126:Brefeldin A; C057416:KT 5720; D000584:Amiloride; D012964:Sodium; D019833:Genistein; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases