Figure 2.

Increased Na-K-ATPase activity and β1 subunit expression in C18 KO mice. (A) Ouabain-sensitive ATPase activity (attributed primarily to Na-K-ATPase activity) was significantly higher (2.3-fold; **P < 0.01) in whole lung membranes from C18 KO (6.3 ± 0.31; n = 3) versus WT mice (2.7 ± 0.12; n = 3). (B) Quantitative RT-PCR (qRT-PCR) analysis of WT and C18 KO whole lung demonstrates relative expression of Na pump subunits (expression level of WT = 1.0). In C18 KO mice, expression of the β1 subunit increased by 61%, whereas α2, α3, and β2 subunits decreased by 56, 53, and 23%, respectively (*P < 0.05). Expression of α1 and β3 subunits remained unchanged. (C) qRT-PCR analysis of Na pump subunit gene expression in freshly isolated AT2 cells from C18 KO mice demonstrates increased expression of β1 and α1 subunits of 30 and 14%, respectively, whereas the α3 subunit was decreased (*P < 0.05) and α2, β2, and β3 subunits were unchanged compared with WT mice. (D) Western blot analysis of whole lung for β1 Na pump subunit demonstrates about 4-fold greater protein levels in C18 KO versus WT mice. ***P < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (E) Western blot analysis of AT2 cell membrane fractions shows significantly higher (2.4-fold) β1 subunit expression in C18 KO versus WT mice. ***P < 0.001. (F) Analysis of β1 mRNA levels in mouse alveolar epithelial cell monolayers (MAECMs) on Day 6 in culture shows no differences between genotypes. (G) Western blot analysis of whole cell lysates from Day 6 MAECMs demonstrates that β1 protein level is unchanged in C18 KO versus WT mice.