Titre du document / Document title

Forskolin-induced cell shrinkage and apical translocation of functional enhanced green fluorescent protein-human αENaC in H441 lung epithelial cell monolayers

Auteur(s) / Author(s)

WOOLLHEAD Alison M. ; BAINES Deborah L. ;

Résumé / 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 aENaC 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 (aC3-5) and full-length (aC3-3) EGFP-haENaC protein. Only the aC3-3 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-haENaC (aC3-3) 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 aC3-3 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-haENaC subunits to the apical membrane and increases amiloride-sensitive Na⁺ transport.

Revue / Journal Title

The Journal of biological chemistry   ISSN 0021-9258   CODEN JBCHA3 

Source / Source

2006, vol. 281, n^o8, pp. 5158-5168 [11 page(s) (article)]

Langue / Language

Anglais

Editeur / Publisher

American Society for Biochemistry and Molecular Biology, Bethesda, MD, ETATS-UNIS  (1905) (Revue)

Localisation / Location

INIST-CNRS, Cote INIST : 3082, 35400015341077.0690