Am. J. Respir. Cell Mol. Biol., Volume 24, Number 3, March, 2001 245-252

Na,K-ATPase Gene Transfer Mitigates an Oxidant-Induced Decrease of Active Sodium Transport in Rat Fetal ATII Cells

Ulrich Thome, Lan Chen, Phillip Factor, Vidas Dumasius, Bruce Freeman, J. Iasha Sznajder, and Sadis Matalon

Departments of Pediatrics and Anesthesiology, University of Alabama at Birmingham, Birmingham, Alabama; and Departments of Medicine, Evanston Northwestern Healthcare and Northwestern University, Chicago, Illinois

We investigated whether adenovirus-mediated transfer of genes encoding for subunits of the Na,K-ATPase increases transepithelial Na⁺ transport in rat fetal distal lung epithelial (FDLE) monolayers and renders them more resistant to hydrogen peroxide injury. FDLE cells, isolated from rat fetuses at a gestational age of 19 to 20 d (22 d = term), were seeded on filters and infected with replication-incompetent human type 5 adenoviruses containing complementary DNAs encoding for rat Na,K-ATPase ₁ or ₁ subunits (ad₁ and ad₁, respectively). Once confluent monolayers were formed, the filters were mounted in Ussing chambers and short circuit currents (I_SC) were measured. Increased levels of ₁ or ₁ subunit proteins after infection with ad₁ and ad₁, respectively, were confirmed by Western blot analysis. Baseline I_SC increased after transfection with 2 plaque-forming units (pfu) of ad₁ from 5.1 ± 0.3 to 6.1 ± 0.3 µA/cm² (mean ± SEM; P < 0.05). Permeabilization of the apical membrane with amphotericin B caused a large increase in I_SC; the ouabain-sensitive component of the amphotericin B-elicited I_SC (ouab_max) was increased from 4.0 ± 0.2 (n = 69) in controls to 4.8 ± 0.2 (n = 15), 5.9 ± 0.3 (n = 53), 6.9 ± 0.4 (n = 25), 7.7 ± 0.9 (n = 16) in monolayers infected with 1, 2, 11, and 22 pfu of ad₁, respectively; transfection with ad₁ had no effect on any measured variables. Further, transfection with ad₁ in comparison to noninfected monolayers resulted in higher baseline and ouab_max I_SC after injury with 500 µM H₂O₂. We conclude that overexpression of the ₁ subunit of the Na,K-ATPase may help maintain normal levels of vectorial Na⁺ transport across ATII cell monolayers in pathologic conditions.

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