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Aquaporin-5 (AQP5) is a water channel protein expressed on the apical surface of alveolar epithelial type I cells in distal rat lung, suggesting a role for AQP5 in regulating alveolar surface liquid tonicity and/or cell volume. We investigated the molecular mechanisms underlying hypertonic induction of AQP5 in primary rat alveolar epithelial cells (AEC). Steady-state levels of AQP5 mRNA and protein were increased by exposure to sorbitol (200 mM in culture fluid) for 24 h. The increase in AQP5 was not accompanied by changes in mRNA half-life. Transduction of mouse lung epithelial (MLE-15) cells and primary rat AEC with lentivirus vectors encoding AQP5-luciferase demonstrated transcriptional activation of the reporter by exposure to hypertonic sorbitol solution. Hybridization of proteins from sorbitol-treated cells to a transcription factor DNA array demonstrated induction of hypoxia-inducible factor-1alpha (HIF-1alpha) by hypertonicity, which was confirmed by quantitative RT-PCR. Cotransfections of AQP5-luciferase with HIF-1alpha and HIF-1beta expression plasmids in MLE-15 cells led to dose-dependent transcriptional enhancement, which was partially abrogated by mutagenesis of putative HIF-1alpha binding sites in the proximal AQP5 promoter. Importantly, hypertonic induction of AQP5 was significantly inhibited by preventing HIF-1alpha induction with small interfering RNA. Hypertonicity induced activation of a transiently transfected vascular endothelial growth factor (VEGF) hypoxia response element-driven luciferase construct and increased expression of endogenous VEGF. These results demonstrate that hypertonic induction of both AQP5 and VEGF is transcriptionally regulated and mediated, at least in part, by HIF-1alpha, suggesting a novel role for HIF-1alpha in modulating cellular adaptive responses to osmotic stress.

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 μM) produced ∼20–30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that α-, β- and γ-subunits of rat epithelial Na+ channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations ([Formula: see text]/[Formula: see text]≈ 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+pump (Na+-K+-ATPase) and rat epithelial Na+channel subunits. Rat AEC were cultivated in minimal defined serum-free 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. Na+channel α-, β-, and γ-subunit and Na+-K+-ATPase α1- and β1-subunit mRNA levels were quantified by Northern analysis. Na+pump α1- and β1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, Iscacross AEC monolayers decreased by ∼60% at 48 h relative to monolayers maintained under normoxic conditions. Na+channel β-subunit mRNA expression was reduced by hyperoxia, whereas α- and γ-subunit mRNA expression was unchanged. Na+pump α1-subunit mRNA was unchanged, whereas β1-subunit mRNA was decreased ∼80% by hyperoxia in parallel with a reduction in β1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-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 β1mRNA 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 Na+pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+pump expression.

We investigated the effects of epidermal growth factor (EGF) on active Na+absorption by alveolar epithelium. Rat alveolar epithelial cells (AEC) were isolated and cultivated in serum-free medium on tissue culture-treated polycarbonate filters. mRNA for rat epithelial Na+channel (rENaC) α-, β-, and γ-subunits and Na+pump α1- and β1-subunits were detected in day 4 monolayers by Northern analysis and were unchanged in abundance in day 5 monolayers in the absence of EGF. Monolayers cultivated in the presence of EGF (20 ng/ml) for 24 h from day 4 to day 5 showed an increase in both α1and β1Na+pump subunit mRNA but no increase in rENaC subunit mRNA. EGF-treated monolayers showed parallel increases in Na+pump α1- and β1-subunit protein by immunoblot relative to untreated monolayers. Fixed AEC monolayers demonstrated predominantly membrane-associated immunofluorescent labeling with anti-Na+pump α1- and β1-subunit antibodies, with increased intensity of cell labeling for both subunits seen at 24 h following exposure to EGF. These changes in Na+pump mRNA and protein preceded a delayed (>12 h) increase in short-current circuit (measure of active transepithelial Na+transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases active Na+resorption across AEC monolayers primarily via direct effects on Na+pump subunit mRNA expression and protein synthesis, leading to increased numbers of functional Na+pumps in the basolateral membranes.