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-β (CaMKK-β). Chelating intracellular Ca 2+ or abrogating CaMKK-β 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-ζ and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-α 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 β-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 β-adrenergic agonists and cAMP.

2؉concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca 2؉ signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.

Background: CO 2 retention and skeletal muscle atrophy occur in patients with lung diseases and are associated with poor clinical outcomes. Results: Hypercapnia leads to AMPK/FoxO3a/MuRF1-dependent muscle fiber size reduction. Conclusion: Hypercapnia activates a signaling pathway leading to skeletal muscle atrophy. Significance: High CO 2 levels directly activate a proteolytic program of skeletal muscle atrophy which is of relevance to patients with lung diseases.

BackgroundIn 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 ∼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 FindingsWe 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 PKCζ which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.ConclusionsOur 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.

Abstract-Alveolar epithelial ␤-adrenergic receptor (␤AR) activation accelerates active Na ϩ transport in lung epithelial cells in vitro and speeds alveolar edema resolution in human lung tissue and normal and injured animal lungs. Whether these receptors are essential for alveolar fluid clearance (AFC) or if other mechanisms are sufficient to regulate active transport is unknown. In this study, we report that mice with no ␤ 1 -or ␤ 2 -adrenergic receptors (␤ 1 AR Ϫ/Ϫ /␤ 2 AR Ϫ/Ϫ ) have reduced distal lung Na,K-ATPase function and diminished basal and amiloride-sensitive AFC. Total lung water content in these animals was not different from wild-type controls, suggesting that ␤AR signaling may not be required for alveolar fluid homeostasis in uninjured lungs. Comparison of isoproterenol-sensitive AFC in mice with ␤ 1 -but not ␤ 2 -adrenergic receptors to ␤ 1 AR Ϫ/Ϫ /␤ 2 AR Ϫ/Ϫ mice indicates that the ␤ 2 AR mediates the bulk of ␤-adrenergic-sensitive alveolar active Na ϩ transport. To test the necessity of ␤AR signaling in acute lung injury, Key Words: alveolar fluid clearance Ⅲ pulmonary edema Ⅲ ␤ 2 -adrenergic receptor Ⅲ adenovirus Ⅲ Na ϩ channel T he combined action of alveolar epithelial Na ϩ channels (ENaCs), the cystic fibrosis transmembrane conductance regulator (CFTR), Na,K-ATPases, and K ϩ channels creates the transepithelial Na ϩ gradient needed for the transit of excess fluid from the alveolar airspace. 1,2 The importance of these proteins to this energy-dependent (ie, active) process is evidenced by data showing that their inhibition reduces the lung's ability to clear excess alveolar fluid [3][4][5][6][7] and that their upregulation confers protection from acute injury. 4,8,9 Despite these extensive investigations, the mechanisms by which these proteins are upregulated in response to excess alveolar fluid (pulmonary edema) are not well resolved.One possible pathway for upregulation of alveolar-active Na ϩ transport is ␤-adrenergic receptor activation. Stimulation of alveolar epithelial ␤ARs by endogenous or exogenous catecholamines accelerates active Na ϩ transport in lung epithelial cells in vitro and in experimental in vivo systems by increasing the expression and/or function of epithelial transport proteins. 10 -12 Thus, this G protein-dependent pathway represents a mechanism by which the lung can alter its physiology to adapt to and protect itself from excess alveolar fluid. What is not known is if ␤AR signaling is essential for the regulation of alveolar active Na ϩ transport or whether other mechanisms (eg, intracellular osmo-, redox-, or chemosensitive regulators) can enhance alveolar active transport to clear pulmonary edema.The present study was structured to define what contribution alveolar epithelial ␤ARs make to active Na ϩ transport in the alveolar epithelium of normal mice and mice with acute lung injury caused by exposure to hyperoxia. Herein, we show that distal lung transport protein function and the lung's ability to clear excess alveolar fluid is highly dependent on Materials an...