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J. Biol. Chem., Vol. 281, Issue 8, 5158-5168, February 24, 2006

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Forskolin-induced Cell Shrinkage and Apical Translocation of Functional Enhanced Green Fluorescent Protein-Human ENaC in H441 Lung Epithelial Cell Monolayers^*

From the Division of Basic Medical Sciences, Ion Channels and Cell Signaling Centre, St. Georges' University of London, Cranmer Terrace, Tooting, London SW17 0RE, United Kingdom

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 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 (C3-5) and full-length (C3-3) EGFP-hENaC protein. Only the 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-hENaC (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 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-hENaC subunits to the apical membrane and increases amiloride-sensitive Na⁺ transport.

Received for publication, September 9, 2005 , and in revised form, December 6, 2005.

^* This work was supported by the Wellcome Trust (Grant 068674/Z/02/Z). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 44-0208-725-0916; Fax: 44-0208-725-2993; E-mail: d.baines{at}sgul.ac.uk.

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