ACELL June 45/6

Borok, Zea, Salim Mihyu, Valentino F. J. Fernandes, Xiao-Ling Zhang, Kwang-Jin Kim, and Richard L. Lubman. KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells. Am. J. Physiol. 276 (Cell Physiol. 45): C1352–C1360, 1999.—We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na1 pump (Na1-K1-ATPase) and rat epithelial Na1 channel subunits. Rat AEC were cultivated in minimal defined serumfree medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2 (normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance (Rt) and short-circuit current (Isc) were determined before and after exposure. Na1 channel a-, b-, and g-subunit and Na1-K1-ATPase a1and b1-subunit mRNA levels were quantified by Northern analysis. Na1 pump a1and b1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, Isc across AEC monolayers decreased by ,60% at 48 h relative to monolayers maintained under normoxic conditions. Na1 channel b-subunit mRNA expression was reduced by hyperoxia, whereas aand g-subunit mRNA expression was unchanged. Na1 pump a1-subunit mRNAwas unchanged, whereas b1-subunit mRNA was decreased ,80% by hyperoxia in parallel with a reduction in b1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na1-K1-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by Isc) relative to normoxic controls. Levels of b1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na1 pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na1 pump expression.