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J. Biol. Chem., Vol. 280, Issue 25, 24181-24187, June 24, 2005

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Differentiation of Epithelial Na⁺ Channel Function

AN IN VITRO MODEL^*

Vadim Shlyonsky, Arnaud Goolaerts¶, Ronald Van Beneden, and Sarah Sariban-Sohraby||

From the Laboratoire de Physiologie et Physiopathologie, Université Libre de Bruxelles, 1070 Bruxelles, Belgium

Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nM dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La³⁺-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (R_t) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na⁺ channel -subunit (-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of - and -ENaC and of 1- and 1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation.

Received for publication, December 8, 2004 , and in revised form, March 18, 2005.

^* This work was supported by funds from the Université Libre de Bruxelles, the Fonds E. DeFay, and the Fonds National de la Recherche Scientifique. 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.

These authors participated equally in this work.

A postdoctoral fellow at the Université Libre de Bruxelles.

^¶ A doctoral fellow at the Université Libre de Bruxelles.

^|| To whom correspondence should be addressed: Laboratoire de Physiologie et Physiopathologie, Campus Erasme CP 604, 808 route de Lennik, 1070 Bruxelles, Belgium. Tel.: 011-322-555-6363; Fax: 011-322-555-4124; E-mail: sohraby{at}ulb.ac.be.

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