ASYMMETRIC EFFECTS OF H2O2 ON ALVEOLAR EPITHELIAL BARRIER PROPERTIES

The effects of H2O2 on active ion transport and resistance to passive solute flow were studied utilizing rat alveolar epithelial cell monolayers cultured on permeable supports. Type II alveolar epithelial cells were plated onto tissue culture-treated polycarbonate membrane filters. The resulting confluent monolayers on days 3 and 4 were mounted in a modified Ussing chamber and bathed on both sides with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer solution. These monolayers have a high transepithelial resistance (>2,000 OMEGA . CM2 ) and actively transport Na+ from apical fluid. H2O2 (0-100 mM) was then delivered to either apical or basolateral fluid. The changes in short-circuit current (I(SC)) and monolayer resistance (R) in response to the exogenous hydroperoxide were measured. To determine the degree of cellular catalase participation in protection against H2O2 injury to the barrier, experiments were repeated in the presence of 20 mM aminotriazole (ATAZ; an inhibitor of catalase) in the same bathing fluid as the hydroperoxide. Results indicated that H2O2 decreased I(SC) and R gradually in a dose-dependent manner. The effective concentration of apical H2O2 at which I(SC) (or R) was decreased by 50% at 1 h (ED50) was approximately 4 mM. However, basolateral H2O2 exposure led to ED50 for I(SC) (and R) of approximately 0.04 mM. Inhibition of cellular catalase yielded ED50 for I(SC) (and R) of approximately 0.4 mM when H2O2 was given apically, while ED50 for basolateral exposure to H2O2 did not change in the presence of ATAZ. The rate of H2O2 consumption in apical and basolateral bathing fluids was the same, while cellular catalase activity rose gradually with time in culture. Our data suggest that basolateral H2O2 may affect directly membrane components (e.g., Na+-K+-adenosinetriphosphatase) located on the basolateral cell surface. Apical H2O2, on the other hand, may be largely degraded by catalase as it passes through the cells before reaching these membrane components. We conclude that alveolar epithelial barrier integrity as measured by I(SC) and R are compromised by H2O2, being relatively sensitive to basolateral (and insensitive to apical) H2O2.