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

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 Isc. Stable expression of green fluorescent protein (GFP)-labeled human alpha ENaC 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 (alpha C3-5) and full-length (alpha C3-3) EGFP-h alpha ENaC protein. Only the alpha C3-3 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-h alpha ENaC (alpha C3-3) 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 alpha C3-3 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-h alpha ENaC subunits to the apical membrane and increases amiloride-sensitive Na+ transport.