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

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

Abstract

Hypercapnia (elevated CO2 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 CO2-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO2 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 CO2-triggered increase in intracellular Ca2+ concentration and Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β). Chelating intracellular Ca2+ or abrogating CaMKK-β function by gene silencing or chemical inhibition prevented the CO2-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-ζ and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-α1 prevented CO2-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 β-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO2 levels are sensed by AECs and that AMPK mediates CO2-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with β-adrenergic agonists and cAMP.

Authors

István Vadász, 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

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Figure 3

AMPK is upstream of PKC-ζ in the CO2-induced signaling cascade in ATII cells.

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AMPK is upstream of PKC-ζ in the CO2-induced signaling cascade in ATII c...
(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.