High CO2 levels impair alveolar epithelial function independently of pH

Abstract

Background: In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO2) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO2 levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO2. The Na,K-ATPase consumes approximately 40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO2 on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function.

Principal findings: We found that short-term increases in pCO2 impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO2, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCzeta which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.

Conclusions: Our data suggest that alveolar epithelial cells, through which CO2 is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO2 levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.

Figure 1. High CO₂ levels impair alveolar epithelial function in rats

. (A) Isolated rat lungs were perfused for 1 h with 40 or 60 mmHg CO₂ with pH_e: 7.4 or pH_e: 7.2 and alveolar fluid reabsorbtion (AFR) was measured as described in the experimental procedures. Graph represents the mean±SEM, (n = 5). (B) Passive movement of Na⁺ (closed bars) and ³H-Mannitol (open bars) was measured as described in detail in the supplementary methods. Graph represents the mean±SEM (n = 5). Differences among groups were not statistically significant. (C) Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of fluorescein isothiocyanate (FITC)-labeled albumin, placed in the perfusate that appeared in the alveolar instillate during the experimental protocol. Graph represents the mean±SEM, (n = 5). Differences among groups were not statistically significant. (D) A three hour experiment was performed in isolated rat lungs. Lungs were perfused for 1h with 40 mmHg CO₂, switched to 60 mmHg CO₂ for the second hour, and back to 40 mmHg CO₂ for the third hour (pH_e: 7.4). Alveolar fluid reabsorption (AFR) was measured as described in the experimental procedures. Graph represents the mean±SEM of 5 independent experiments. Bars in panels A, B and C represent the mean from different groups of animals. Bars in panel D represent the mean of single samples from the same group of animals. pH_e: extracellular pH. *p<0.05; **p<0.01.

Figure 2. Na,K-ATPase function is impaired in rat lungs exposed to hypercapnic acidosis.

(A) Basolateral membranes (BLM) were purified from the peripheral lung tissue of rat lungs exposed to 40 mmHg CO₂ (pH_e: 7.4) or 60 mmHg CO₂ (pH_e: 7.2), and Na,K-ATPase activity was measured as [γ-³²P]ATP hydrolysis. Graph represents the mean±SEM, (n = 3). (B) BLM and total membranes were purified from the peripheral lung tissue of rat lungs treated as (A), and Na,K-ATPase protein abundance was assessed by Western blot. Graph represents the mean±SEM, (n = 3). Representative blots of Na,K-ATPase α₁-subunit at the BLM and total membrane protein abundance are shown. pH_e: extracellular pH. * p<0.05.

Figure 3. High CO₂ levels impair Na, K-ATPase activity independently of pH.

(A) ATII cells were exposed to 40, 60, 80 and 120 mmHg CO₂ with extracellular pH (pH_e): 7.4 or to 40 mmHg CO₂ with pH_e: 7.2 for 30 min, and Na,K-ATPase activity was measured as [γ-³²P]ATP hydrolysis. Graph represents the mean±SEM, (n = 5). (B) ATII cells were treated as described in (A) and the Na,K-ATPase protein abundance at the plasma membrane (PM) was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean±SEM, (n = 5). Representative blots of Na,K-ATPase α₁-subunit at the PM and total protein abundance are shown. (C) Live cell imaging of GFPα₁-A549. Cells were exposed to 40 mmHg CO₂ (pH_e: 7.4) for 10 min (left panels) and then switched to 120 mmHg CO₂ (pH_e: 7.4) (right, upper panel) or to 40 mmHg CO₂ (pH_e: 7.2) (right, lower panel) for 30 min. White arrows indicate the plasma membrane Na,K-ATPase. (D) Live cell imaging of GFPα₁-A549. Cells were exposed to 40 mmHg CO₂ (pH_e: 7.4) for 10 min (left panel), switched to 120 mmHg CO₂ (pH_e: 7.4) (middle panel) for 30 min and switched back to 40 mmHg CO₂ (pH_e: 7.4) for 60 min (left panel). White arrows indicate the plasma membrane Na,K-ATPase * p<0.05, ** p<0.01.

Figure 4. Effects of high CO₂ and extracellular acidosis on intracellular pH.

Intracelluar pH (pH_i) was measured in real time as the change in fluorescence intensity of ATII cells loaded with BCECF/AM and exposed approximately 3 min to 40 mmHg, and then for the indicated time to 60, 80 and 120 mmHg CO₂ with pH_e: 7.4, or 40 mmHg with pH_e: 7.2. pH_e: extracellular pH.

Figure 5. Role of PKCζ in CO₂-induced Na,K-ATPase α₁-subunit endocytosis.

(A) ATII cells were exposed for the indicated times to 40 or 120 mmHg CO₂ (pH_e: 7.4), cytosolic and 1% Triton X-100 soluble fractions were isolated, and translocation of different PKC isoforms was determined by Western blot with specific antibodies. Representative blots for PKCα, PKCε and PKCζ are shown (n = 3). (B) ATII cells were incubated with vehicle, 5 µM PKCε inhibitory peptide or 0.1 µM PKCζ inhibitory peptide 1 h prior to being exposed to 40 or 120 mmHg CO₂ (pH_e: 7.4) for 30 min. Na,K-ATPase protein abundance at the PM was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean±SEM, (n = 5). Representative blots of Na,K-ATPase α₁-subunit at the PM and total protein abundance are shown. (C) A549 cells expressing an empty vector or a DN-PKCζ were exposed to 40 or 120 mmHg CO₂ (pH_e: 7.4) for 30 min. The Na,K-ATPase protein abundance at the PM was determined as above. Graph represents the mean±SEM, (n = 5). Representative blots of Na,K-ATPase α₁-subunit at the PM and total protein abundance are shown. (D) Isolated rat lungs from rats infected with Sham-surfactant, with null adenoviral vector (Ad-null), and adenoviral vector with DN PKCζ construct (Ad-DN-PKCζ) were perfused for 1 h with 40 mmHg CO₂ (pH_e: 7.4) or with 60 mmHg CO₂ (pH_e: 7.2), and AFR was measured as described in the Methods section. Graph represents the mean±SEM, (n = 5). (E) Lungs from rats infected with Sham, Ad-null and Ad-DN-PKCζ were thoroughly rinsed with ice-cold PBS, tissue was homogenized, and the abundance of PKCζ protein abundance was determined by Western blot. Representative Western blots of PKCζ and actin (loading control) are shown. (F) Lung tissues from rats infected with Sham, Ad-null and Ad-DN-PKCζ were thoroughly rinsed with ice-cold PBS and fixed in 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining was performed as described in the Online Data Supplement. Magnification x40. *p<0.05, **p<0.01.

Figure 6. PKCζ phosphorylates the Na,K-ATPase α₁-subunit at Ser 18.

(A) In vitro “back phosphorylation” assay was performed (as described in the methods section) on the immunoprecipitated Na,K-ATPase α₁-subunit from GFPα1-A549 cells exposed to 40 or 120 mmHg CO₂ (pH_e: 7.4) for 10 min in the presence or absence of PKCζ inhibitory peptide (0.1 µM, 1 h). Upper panel shows a representative autoradiography. Lower panel depicts a representative Western blot (n = 3; p<0.05 when comparing 40 mmHg vs 120 mmHg). (B) A549 cells expressing the rat GFPα₁-subunit Na,K-ATPase (WT) or the rat GFP-S18A α₁-subunit (S18A) were exposed to 40 or 120 mmHg CO₂ (pH_e: 7.4) for 30 min. The Na,K-ATPase protein abundance at the plasma membrane (PM) was determined by biotin-streptavidin pull down and subsequent Western blot. Graph represents the mean±SEM, (n = 5). Representative blots of Na,K-ATPase α₁-subunit at the PM and total protein abundance are shown. (C) Live cell imaging of A549 cells expressing the rat GFPα₁-subunit Na,K-ATPase (WT) or the rat GFP-S18Aα₁-subunit (S18A). Cells were exposed to 40 mmHg CO₂ (pH_e: 7.4) for 10 min (left panels) and then switched to 120 mmHg CO₂ (pH_e: 7.4) for 30 min (right panels). White arrows indicate the plasma membrane Na,K-ATPase. * p<0.05.