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.

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.

The development of nonviral methods for efficient gene transfer to the lung is highly desired for the treatment of several pulmonary diseases. We have developed a noninvasive procedure using electroporation to transfer genes to the lungs of rats. Purified plasmid (100-600 microg) was delivered to the lungs of anesthetized rats through an endotracheal tube, and a series of square-wave pulses were delivered via electrodes placed on the chest. Relatively uniform gene expression was observed in multiple cell types and layers throughout the lung, including airway and alveolar epithelial cells, airway smooth muscle cells, and vascular endothelial cells, and this finding was dose- and pulse length-dependent. Most important, no inflammatory response was detected. To demonstrate efficacy of this approach, the beta1 subunit of the Na(+),K(+)-ATPase was transferred to the lungs of rats with or without electroporation, and 3 days later, alveolar fluid clearance was measured. Animals electroporated with the beta1 subunit plasmid showed a twofold increase in alveolar fluid clearance and Na(+),K(+)-ATPase activity as compared with animals receiving all other plasmids, with or without electroporation. These results demonstrate that electroporation is an effective method to increase clearance by introducing therapeutic genes (Na(+),K(+)-ATPase) into the rat lung.

Using LAURDAN spectral imaging and spectral phasor analysis we concurrently studied the growth and hydration state of subcellular organelles (Lamellar Body-like, LB-like) from live A549 lung cancer cells at different post-confluence days. Our results reveal a time dependent two-step process governing the size and hydration of these intracellular LB-like structures. Specifically, a first step (days 1 to 7) is characterized by an increase in their size, followed by a second one (days 7 to 14) where the organelles display a decrease in their global hydration properties. Interestingly, our results also show that their hydration properties significantly differ from those observed in well-characterized artificial lamellar model membranes, challenging the notion that a pure lamellar membrane organization is present in these organelles at intracellular conditions. Finally, these LB-like structures show a significant increase in their hydration state upon secretion, suggesting a relevant role of entropy during this process.

Elevated CO2 levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO2 levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO2 signaling in mammals, diptera and nematodes.

Hypoxia has been shown to cause lung edema and impair lung edema clearance. In the present study, we exposed isolated rat lungs to pO 2 ‫؍‬ 40 mm Hg for 60 min or rats to 8% O 2 for up to 24 h and then measured changes in alveolar fluid reabsorption (AFR) and Na,K-ATPase function. Low levels of oxygen severely impaired AFR in both ex vivo and in vivo models. The decrease in AFR was associated with a decrease in Na,K-ATPase activity and protein abundance in the basolateral membranes from peripheral lung tissue of hypoxic rats. ␤-Adrenergic agonists restored AFR in rats exposed to 8% O 2 (from 0.02 ؎ 0.07 ml/h to 0.59 ؎ 0.03 ml/h), which was associated with parallel increases in Na,K-ATPase protein abundance in the basolateral membrane. Hypoxia is associated with increased production of reactive oxygen species. Therefore, we examined whether overexpression of SOD2, manganese superoxide dismutase, would prevent the hypoxia-mediated decrease in AFR. Spontaneously breathing rats were infected with a replication-deficient human type 5 adenovirus containing cDNA for SOD2. An otherwise identical virus that contained no cDNA was used as a control (Adnull). Hypoxic Adnull rats had decreased rates of AFR (0.12 ؎ 0.1 ml/h) as compared with hypoxic AdSOD2 and normoxic control rats (0.47 ؎ 0.04 ml/h and 0.49 ؎ 0.02 ml/h, respectively), with parallel changes in Na,K-ATPase.Severe hypoxia can occur during ascent to high altitude (1) and in patients with acute respiratory distress syndrome and pulmonary edema. One of the primary defense mechanisms in the lung against alveolar fluid accumulation is the active transport of sodium out of the air spaces, which generates a transepithelial osmotic gradient that leads to alveolar fluid reabsorption (AFR).2 Sodium enters the apical membrane of alveolar epithelial cells through amiloride-sensitive Na ϩ channels (2, 3) and is then transported out across the basolateral membrane by the ouabain-inhibitable Na,K-ATPase (4 -7). Hypoxia has been shown to impair AFR and may contribute to alveolar fluid accumulation (8, 9). However, the mechanisms by which hypoxia impairs AFR and alveolar epithelial sodium transport proteins has not been fully elucidated.A mechanism by which hypoxia might impair AFR is by altering the function of either apical epithelial sodium channels and/or basolateral Na,K-ATPase proteins. Several in vitro studies using cultured alveolar epithelial cells have demonstrated that exposure to hypoxia results in the decrease in epithelial sodium channels and Na,K-ATPase protein abundance (10 -12), which was reversed upon reoxygenation. Other investigators have reported various mechanisms associated with the decrease in alveolar fluid reabsorption in animals exposed to hypoxia in vivo (9,11,13).In the current study, we provide evidence that exposure to hypoxia results in decreased Na,K-ATPase activity and protein abundance at the plasma membrane, which contributes to a decrease in alveolar fluid reabsorption in both in vivo and ex vivo models of hypoxia. These data suggest that (a...

a b s t r a c tHypercapnia has been shown to impair alveolar fluid reabsorption (AFR) by decreasing Na,K-ATPase activity. Extracellular signal-regulated kinase pathway (ERK) is activated under conditions of cellular stress and has been known to regulate the Na,K-ATPase. Here, we show that hypercapnia leads to ERK activation in a time-dependent manner in alveolar epithelial cells (AEC). Inhibition of ERK by U0126 or siRNA prevented both the hypercapnia-induced Na,K-ATPase endocytosis and impairment of AFR. Moreover, ERK inhibition prevented AMPK activation, a known modulator of hypercapniainduced Na,K-ATPase endocytosis. Accordingly, these data suggest that hypercapnia-induced Na,K-ATPase endocytosis is dependent on ERK activation in AEC and that ERK plays an important role in hypercapnia-induced impairment of AFR in rat lungs.