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. 2008 Feb;118(2):752-62.
doi: 10.1172/JCI29723.

AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis

István Vadász  1 , Laura A Dada, Arturo Briva, Humberto E Trejo, Lynn C Welch, Jiwang Chen, Péter T Tóth, Emilia Lecuona, Lee A Witters, Paul T Schumacker, Navdeep S Chandel, Werner Seeger, Jacob I Sznajder
Affiliations

AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis

István Vadász et al. J Clin Invest. 2008 Feb.

Abstract

Hypercapnia (elevated CO(2) levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO(2)-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO(2) levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO(2)-triggered increase in intracellular Ca(2+) concentration and Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta function by gene silencing or chemical inhibition prevented the CO(2)-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(1) prevented CO(2)-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a beta-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO(2) levels are sensed by AECs and that AMPK mediates CO(2)-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with beta-adrenergic agonists and cAMP.

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Figures

Figure 1
Figure 1. High CO2 levels activate AMPK in a concentration- and time-dependent manner in ATII cells.
(A) ATII cells were exposed to 40, 60, 80, and 120 mmHg CO2 with an extracellular pH (pHe) of 7.4 (white and black bars) or to 40 mmHg CO2 with a pHe of 7.2 (gray bar) for 5 min, and the phosphorylation of AMPK at Thr172 (pAMPK-α) and the total amount of AMPK-α was measured by Western blot analysis. Graph represents the pAMPK/AMPK ratio. Values are expressed as mean ± SEM. n = 4. Representative Western blots of pAMPK-α and total AMPK-α. (B) ATII cells were treated with 40 mmHg CO2 (white bar) for 5 min or with 120 mmHg CO2 for 15 s to 5 min (black bars) at a pHe of 7.4. Graph represents the pAMPK/AMPK ratio. Values are expressed as mean ± SEM. n = 4. Representative Western blots of pAMPK-α and total AMPK-α. *P < 0.05; **P < 0.01.
Figure 2
Figure 2. Activation of AMPK is both necessary and sufficient to promote Na,K-ATPase endocytosis in ATII cells.
(A) ATII cells were exposed to 40 or 120 mmHg CO2 for 30 min in the presence or absence of compound C (20 μM, 30 min preincubation). Na,K-ATPase at the plasma membrane was determined by biotin-streptavidin pull-down and Western blot. Representative Western blots of Na,K-ATPase-α1 at the plasma membrane (PM) and total protein abundance are shown. (B) ATII cells were infected with Ad-null or HA-tagged Ad-DN–AMPK-α1 and were exposed to 40 or 120 mmHg CO2 for 30 min. Na,K-ATPase at the plasma membrane was determined as described above. Mean ± SEM. n = 4. Representative Western blots of the Na,K-ATPase-α1 at the plasma membrane, total protein abundance, and level of HA-tagged AMPK expression are shown. (C) ATII cells were treated with 2 mM AICAR for 30 or 60 min or with vehicle for 60 min, and the Na,K-ATPase at the plasma membrane was determined as above. Representative Western blots of Na,K-ATPase-α1 at the plasma membrane and total protein abundance are shown. (D) ATII cells were infected with Ad-null (50 pfu/cell) or a GFP-tagged Ad-CA–AMPK-α (2, 10, or 50 pfu/cell), and the amount of Na,K-ATPase was determined as described. Representative Western blots of Na,K-ATPase-α1 at the plasma membrane, total protein abundance, and expression GFP CA-AMPK are shown. Typical phase-contrast (PC) and GFP images of infected cells are shown. Mean ± SEM. n = 4. *P < 0.05; **P < 0.01.
Figure 3
Figure 3. AMPK is upstream of PKC-ζ in the CO2-induced signaling cascade in ATII cells.
(A) ATII cells were exposed to 40 (Ctrl for 15 min) or 120 mmHg CO2 for the indicated times. PKC-ζ was immunoprecipitated and incubated with MBP and [γ–32P]ATP. A representative autoradiograph of the phosphorylated MBP and a Western blot of the immunoprecipitated PKC-ζ are shown. The right lane in the autoradiograph shows the result when the kinase assay was performed in the absence of MBP. n = 4 (B) ATII cells were exposed to 40 or 120 mmHg CO2 for 5 min in the presence or absence of compound C. PKC-ζ translocation was assessed as described in Methods. Representative Western blots for PKC-ζ and the loading control E-cadherin (a membrane protein) are shown. (C) ATII cells were infected with Ad-null or HA-tagged Ad-DN–AMPK-α1 and exposed to 40 or 120 mmHg CO2 for 5 min. PKC-ζ translocation was determined as described above. Representative Western blots for PKC-ζ and E-cadherin are shown. (D) ATII cells were treated with 2 mM AICAR for the indicated times or with vehicle for 30 min, and translocation of PKC-ζ was determined as described above. Representative Western blots for PKC-ζ and E-cadherin are illustrated. (E) ATII cells were exposed to 40 or 120 mmHg CO2 for 5 min in the presence or absence of bisindolylmaleimide I (Bis; 10 μM, 30 min preincubation) or a myristoylated peptide inhibitor of PKC-ζ (15 μM, 30 min preincubation). pAMPK-α and total AMPK-α were determined by Western blot. Graph represents the pAMPK/AMPK ratio. Representative Western blots of pAMPK-α and total AMPK-α are shown. (F) ATII cells were infected with Ad-null or Ad-DN–PKC-ζ and exposed to 40 or 120 mmHg CO2 for 5 min. pAMPK-α and total AMPK-α were measured by Western blot. Graph represents the pAMPK/AMPK ratio. Representative Western blots of pAMPK-α and total AMPK-α and PKC-ζ in whole cell lysates are shown. Mean ± SEM. n = 4. *P < 0.05; **P < 0.01.
Figure 4
Figure 4. CO2-induced activation of AMPK is Ca2+ dependent.
(A) A representative trace of Ca2+ influx into ATII cells exposed to high CO2 levels is illustrated. Fura-2–loaded cells were initially perfused with media containing 40 mmHg CO2, and then perfusion was switched to 120 mmHg CO2 while pHe was maintained at 7.4. Changes in [Ca2+]i are expressed as the F340/F380 ratio. n = 3. (B) ATII cells were exposed to 40 (white bars) or 120 (black bars) mmHg CO2 (pHe 7.4) for 5 min in the presence or absence of BAPTA-AM (20 μM, 20-min preincubation). pAMPK-α and total AMPK-α were determined by Western blot. Graph represents the pAMPK/AMPK ratio. Values are expressed as mean ± SEM. n = 4. **P < 0.01. Representative Western blots of pAMPK-α and total AMPK-α are shown.
Figure 5
Figure 5. CO2-induced activation of AMPK is mediated by CaMKK-β in ATII and A549 cells.
(A and B) ATII (A) and A549 cells (B) were exposed to 40 (white bars) or 120 (black bars) mmHg CO2 (pHe 7.4) for 5 min in the presence or absence of STO-609 (20 μg/ml, 30 min preincubation). pAMPK-α and total AMPK-α were determined by Western blot. Graphs represent pAMPK/AMPK ratios. Values are expressed as mean ± SEM. n = 4. **P < 0.01; ###P < 0.001 compared with untreated cells exposed to 40 or 120 mmHg CO2. (C) Expression levels of LKB1 and CaMKK-β in rat ATII and A549 cells are depicted in representative Western blots. Equal amounts of protein were loaded in each lane. Homogenates of rat kidney and rat cerebrum are shown on the left lane as positive controls for LKB1 and CaMKK-β, respectively. (D) A549 cells were transfected with siRNA against CaMKK-β or scrambled siRNA, and 48 h later cells were exposed to 40 (white bars) or 120 (black bars) mmHg CO2 (pHe 7.4) for 5 min. pAMPK-α and total AMPK-α were determined by Western blot. Graph represents the pAMPK/AMPK ratio. Values are expressed as mean ± SEM. n = 4. **P < 0.01 compared with control values; ###P < 0.001 compared with both 40 and 120 mmHg CO2 controls.
Figure 6
Figure 6. AMPK mediates the hypercapnia-induced impairment of AFR in rat lungs.
(A) Isolated rat lungs were perfused for 1 h with 40 mmHg CO2 (pHe 7.4; white bars) or with approximately 60 mmHg CO2 (pHe 7.2; black bars) in the presence or absence of compound C (20 μM, 30 min preincubation), and AFR was measured as described in the supplemental material. Bars represent the mean ± SEM. n = 5. **P < 0.01. (B) Passive fluxes of 22Na+ (black bars) and 3H-mannitol (white bars) were measured as described in the supplemental material. Data represent the mean ± SEM. n = 5. (C) Isolated rat lungs from animals infected with surfactant (sham), Ad-null, or Ad-DN–AMPK-α1 were perfused for 1 h with 40 mmHg CO2 (pHe 7.4; white bars) or with 60 mmHg CO2 (pHe 7.2; black bars), and AFR was measured as described in detail in the supplemental material. Bars represent the mean ± SEM. n = 5. **P < 0.01. (D) Passive fluxes of 22Na+ (black bars) and 3H-mannitol (white bars) were measured as described in the supplemental material. Data represent the mean ± SEM. n = 5. (E) H&E staining, phase contrast (PC), and GFP images after HA immunohistochemistry of peripheral lung tissues from sham and Ad-null– and Ad-DN–AMPK-α1–infected rats are shown. (F) Representative Western blots of HA-tagged AMPK and actin (as a loading control) are shown from peripheral lung tissue homogenates from sham and Ad-null– and Ad-DN–AMPK-α1–infected rats.
Figure 7
Figure 7. Long-term exposure of rats to hypercapnia results in sustained impairment of AFR.
(A) Rats were maintained in room-temperature air (Ctrl) or at 10% CO2 and 21% O2 for 3 or 7 days and compared with isolated rat lungs that were perfused for 1 h with approximately 60 mmHg CO2, and AFR was measured as described in the supplemental material. Bars represent the mean ± SEM. n = 5. **P < 0.01. (B) Passive fluxes of 22Na+ (black bars) and 3H-mannitol (white bars) were measured as described above. Data represent the mean ± SEM. n = 5. (C) ATII cells were exposed to 40 (white bars) or 120 (black bars) mmHg CO2 (pHe 7.4) for the indicated times. The Na,K-ATPase protein abundance at the plasma membrane was determined by biotin-streptavidin pull-down and subsequent Western blot analysis. Bars represent the mean ± SEM. n = 4. **P < 0.01. Representative Western blots of Na,K-ATPase α1-subunit at the plasma membrane and total protein abundance are shown. White line indicates that lanes were run on the same gel but were noncontiguous.
Figure 8
Figure 8. The hypercapnia-induced impairment of AFR in rat lung is ameliorated by isoproterenol and 8Br.
(A) Isolated rat lungs were perfused for 1 h with 40 mmHg CO2 in the absence (Ctrl) and then for 1 h in the presence of isoproterenol (1 μM, ISO), after which perfusion was switched to approximately 60 mmHg CO2 in the continuous presence of isoproterenol, and AFR was measured as described in the supplemental material. Bars represent the mean ± SEM. n = 5. **P < 0.01. (B) Isolated rat lungs were perfused for 1 h with 40 mmHg CO2 in the absence and then for 1 h in the presence of 8Br (100 μM), after which perfusion was switched to approximately 60 mmHg CO2 in the continuous presence of 8Br, and AFR was measured as indicated above. Bars represent the mean ± SEM. n = 5. **P < 0.01. (C) Isolated rat lungs were perfused for 1 h with 40 mmHg CO2, and then perfusion was switched to approximately 60 mmHg for 1 h in the absence and then for 1 h in the presence of isoproterenol (1 μM). AFR was measured as described in the supplemental material. Bars represent the mean ± SEM. n = 5. **P < 0.01.
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