Figure 1.

High CO₂ exposure increases the production of cyclic adenosine monophosphate (cAMP) via soluble adenylyl cyclase (sAC) in alveolar epithelial cells, which is necessary for Na,K-ATPase endocytosis. (A) Rat alveolar type II (ATII), human A549, and rat RLE-6TN cells were exposed to 40 mm Hg Pco₂ (CT) or 120 mm Hg Pco₂ (CO₂) for 1 minute, and cAMP production was measured by immunoassay (n = 4). (B) Graph shows a representative fluorescence resonance energy transfer imaging (FRET) tracing in A549 cells expressing the guanine nucleotide exchange factor for the small G protein Rap-1 (Epac-1) sensor and exposed to CT or CO₂ in the presence or absence of 5 μM 2-hydroxyestradiol (2HE). An increase in the ratio corresponds to an increase in cAMP production. Arrow indicates when cells were exposed to high CO₂ (n = 4). (C) ATII cells were preincubated for 30 minutes with vehicle or with 5 μM 2HE and were exposed to CT or CO₂ for 1 minute, and cAMP production was measured by immunoassay (n = 5). (D) RLE cells were transfected with scrambled small interfering (si)RNA (si-scr) or siRNA for sAC (si-sAC) and were exposed to CT or CO₂ for 1 minute, and cAMP production was measured by immunoassay (n = 3). (E) Graph represents the relative abundance of the Na,K-ATPase α1 subunit at the plasma membrane in ATII cells preincubated for 30 minutes with vehicle or 5 μM 2HE and exposed to CT or CO₂ for 30 minutes. Below: A representative Western blot shows the abundance of the Na,K-ATPase α1 subunit at the plasma membrane (PM) and cell lysate (CL) (n = 5). (F) Graph represents the relative abundance of the Na,K-ATPase α1 subunit at the plasma membrane in RLE cells transfected with si-scr or si-sAC and exposed to CT or CO₂ for 30 minutes. Below: A representative Western blot shows the abundance of the Na,K-ATPase α1 subunit at the plasma membrane (PM) and cell lysate (CL) and sAC knockdown (n = 3). *P < 0.05. **P < 0.01.