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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.

-Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of ␣-smooth muscle actin (␣-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of ␣-SMA and vimentin was prevented in mitochondria-deficient 0 cells, which are incapable of ROS production during hypoxia. CoCl2 and dimethyloxaloylglycine, two compounds that stabilize hypoxiainducible factor (HIF)-␣ under normoxia, failed to induce ␣-SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1␣ did not induce ␣-SMA. However, loss of HIF-1␣ or HIF-2␣ abolished induction of ␣-SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-␤1, and preincubation of AEC with SB431542, an inhibitor of the TGF-␤1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-␤1 dependent. Furthermore, both ROS and HIF-␣ were necessary for hypoxia-induced TGF-␤1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-␤1 signaling. alveolar epithelial cells; pulmonary fibrosis; transforming growth factor-␤1 EPITHELIAL-MESENCHYMAL TRANSITION (EMT) is a cellular process during which epithelial cells acquire mesenchymal properties while losing cell-cell interactions and apicobasal polarity (33,44). EMT is characterized by changes in cell morphology and acquisition of mesenchymal markers such as ␣-smooth muscle actin (␣-SMA) and vimentin as well as loss of epithelial makers, including E-cadherin (53). Transforming growth factor (TGF)-␤1 is considered to be the prototypical cytokine for the induction of EMT (53). Active TGF-␤1 binds to the transmembrane serine-threonine kinase receptor II and receptor I and activates Smad-mediated transcription of target genes, including ␣-SMA and vimentin, which leads to EMT (33,53,54). TGF-␤1 is reported to induce EMT in renal proximal tubular epithelial cells, lens epithelial cells, and, most recently, alveolar epithelial cells (AEC) (19,23,40,48,55).AEC perform many tasks necessary for normal alveolus functioning, including surfactant protein production and fluid and ion transport (17, 57). Recent evidence suggests that AEC may undergo EMT, contributing to the pathogenesis of pulmonary fibrosis (26,49).AEC are exposed to hypoxia in human lung diseases, including acute lung injury and pulmonary fibrosis (20, 41, 57). It has been described that during hypoxia, mitochondria increase the production of reactive oxy...

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.

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase C (PKC)-mediated phosphorylation of the Na,K-ATPase ␣ subunit. Here, we report that hypoxia leads to the phosphorylation of 5-AMPactivated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK ␣ subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient 0 -A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKC translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK ␣ phosphorylates PKC on residue Thr410 within the PKC activation loop. Importantly, the activation of AMPK ␣ was necessary for hypoxia-induced AMPK-PKC binding in alveolar epithelial cells. The overexpression of T410A mutant PKC prevented hypoxia-induced Na,KATPase endocytosis, confirming that PKC Thr410 phosphorylation is essential for this process. PKC activation by AMPK is isoform specific, as small interfering RNA targeting the ␣1 but not the ␣2 catalytic subunit prevented PKC activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK ␣1 isoform, which binds and directly phosphorylates PKC at Thr410, thereby promoting Na,K-ATPase endocytosis.When exposed to low oxygen levels (hypoxia), cells develop adaptative strategies to maintain adequate levels of ATP (21). These strategies include increasing the efficiency of energyproducing pathways, mostly through anaerobic glycolysis, while decreasing energy-consuming processes such as Na,K-ATPase activity (30). Alveolar hypoxia occurs in many respiratory disorders, and it has been shown to decrease epithelial active Na ϩ transport, leading to impaired fluid reabsorption (37,41,42). Active Na ϩ transport and, thus, alveolar fluid reabsortion are effected mostly via apical sodium channels and the basolateral Na,K-ATPase (32, 38, 42). We have reported previously that hypoxia inhibits Na,K-ATPase activity by promoting its endocytosis from the plasma membrane by a mechanism that requires the generation of mitochondrial reactive oxygen species (ROS) and the phosphorylation of the Na,K-ATPase ␣ subunit at Ser18 by protein kinase C (PKC) (8, 9).The 5Ј-AMP-activated protein kinase (AMPK) is a heterotrimeric Ser/Thr kinase composed of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. Both isoforms of the AMPK catalytic subunit (␣1 and ␣2) form complexes with noncatalytic subunits. The ␣1 subunit is ubiquitously expressed, whereas the ␣2 subunit isoform is expressed predominantly in tissues like the liver, heart, and skeletal muscle (36). The ␣1 and ␣2 subunit isoforms have ϳ90% homology in their N-terminal ca...

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 microM DA or a dopaminergic D(1) agonist, fenoldopam, but not with the dopaminergic D(2) agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase alpha1 and beta1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-delta, or isozyme-specific inhibitor peptides for PKC-delta or PKC-epsilon inhibited the DA-mediated increase in Na,K-ATPase. PKC-delta and PKC-epsilon, but not PKC-alpha or -beta, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-delta- and PKC-epsilon-specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-delta and PKC-epsilon.