Previous studies have suggested that alveolar Na,K-ATPases play an important role in active Na ϩ transport and lung edema clearance. We reasoned that overexpression of Na,K-ATPase subunit genes could increase Na,K-ATPase function in lung epithelial cells and edema clearance in rat lungs. To test this hypothesis we produced replication deficient human type 5 adenoviruses containing cDNAs for the rat ␣ 1 and ␤ 1 Na,K-ATPase subunits (adMRCMV ␣ 1 and adMRCMV ␤ 1 , respectively). As compared to controls, adMRCMV ␤ 1 increased ␤ 1 subunit expression and Na,KATPase function by 2.5-fold in alveolar type 2 epithelial cells and rat airway epithelial cell monolayers. No change in Na,K-ATPase function was noted after infection with adMRCMV ␣ 1 . Rat lungs infected with adMRCMV ␤ 1 , but not adMRCMV ␣ 1 , had increased ␤ 1 protein levels and lung liquid clearance 7 d after tracheal instillation. Alveolar epithelial permeability to Na ϩ and mannitol was mildly increased in animals infected with adMRCMV ␤ 1 and a similar Escherichia coli lacZ -expressing virus. Our data shows, for the first time, that transfer of the ␤ 1 Na,K-ATPase subunit gene augments Na,K-ATPase function in epithelial cells and liquid clearance in rat lungs. Conceivably, overexpression of Na,K-ATPases could be used as a strategy to augment lung liquid clearance in patients with pulmonary edema. (

Abstract-The alveolar epithelium is composed of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells, which represent Ϸ95% and Ϸ5% of the alveolar surface area, respectively. Lung liquid clearance is driven by the osmotic gradient generated by the Na,K-ATPase. AT2 cells have been shown to express the ␣1 Na,K-ATPase. We postulated that AT1 cells, because of their larger surface area, should be important in the regulation of active Na ϩ transport. By immunofluorescence and electron microscopy, we determined that AT1 cells express both the ␣1 and ␣2 Na,K-ATPase isoforms. In isolated, ouabain-perfused rat lungs, the ␣2 Na,K-ATPase in AT1 cells mediated 60% of the basal lung liquid clearance. The ␤-adrenergic agonist isoproterenol increased lung liquid clearance by preferentially upregulating the ␣2 Na,K-ATPase protein abundance in the plasma membrane and activity in alveolar epithelial cells (AECs). Rat AECs and human A549 cells were infected with an adenovirus containing the rat Na,K-ATPase ␣2 gene (Ad␣2), which resulted in the overexpression of the ␣2 Na,K-ATPase protein and caused a 2-fold increase in Na,K-ATPase activity. Spontaneously breathing rats were also infected with Ad␣2, which increased ␣2 protein abundance and resulted in a Ϸ250% increase in lung liquid clearance. These studies provide the first evidence that ␣2 Na,K-ATPase in AT1 cells contributes to most of the active Na ϩ transport and lung liquid clearance, which can be further increased by stimulation of the ␤-adrenergic receptor or by adenovirus-mediated overexpression of the ␣2 Na,K-ATPase.

Pulmonary edema is cleared via active Na(+) transport by alveolar epithelial Na(+)/K(+)-ATPases and Na(+) channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na(+)/K(+)-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na(+)/K(+)-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 x 10(9) PFU of recombinant adenoviruses containing rat alpha(1) and beta(1) Na(+)/K(+)-ATPase subunit cDNAs (adalpha(1) and adbeta(1), respectively) to rat lungs 7 days prior to exposure to 100% O(2) for 64 hr. As compared with controls and ad alpha(1), AFC in the adbeta(1) rats was increased by >300%. Permeability for large solutes was less in the ad beta(1) than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adbeta(1) group. Survival through 14 days of hyperoxia was 100% in the adbeta(1) group but was not different from hyperoxic controls in animals given adalpha(1). Our data show that overexpression of a beta(1) Na(+)/K(+)-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na(+)/K(+)-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.

β-Adrenergic agonists have been reported to increase lung liquid clearance by stimulating active Na+ transport across the alveolar epithelium. We studied mechanisms by which β-adrenergic isoproterenol (Iso) increases lung liquid clearance in isolated perfused fluid-filled rat lungs. Iso perfused through the pulmonary circulation at concentrations of 10−4 to 10−8 M increased lung liquid clearance compared with that of control lungs ( P < 0.01). The increase in lung liquid clearance was inhibited by the β-antagonist propranolol (10−5 M), the Na+-channel blocker amiloride (10−4 M), and the antagonist of Na-K-ATPase, ouabain (5 × 10−4 M). Colchicine, which inhibits cell microtubular transport of ion-transporting proteins to the plasma membrane, blocked the stimulatory effects of Iso on active Na+ transport, whereas the isomer lumicolchicine, which does not affect cell microtubular transport, did not inhibit Na+ transport. In parallel with these changes, the Na-K-ATPase α1-subunit protein abundance and activity increased in alveolar type II cells stimulated by 10−6 M Iso. Colchicine blocked the stimulatory effect of Iso and the recruitment of Na-K-ATPase α1-protein to the basolateral membrane of alveolar type II cells. Accordingly, Iso increased active Na+ transport and lung liquid clearance by stimulation of β-adrenergic receptors and probably by upregulation of apical Na+ channels and basolateral Na-K-ATPase mechanisms. Recruitment from intracellular pools and microtubular transport of Na+pumps to the plasma membrane participate in β-adrenergic stimulation of lung liquid clearance in rat lungs.