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.

Lung liquid clearance, epithelial permeability for Na+, mannitol and albumin, as well as Na,K-ATPase activity in alveolar type 2 (AT2) cells were studied during the acute and the recovery phase of hyperoxic lung injury. Rats exposed to 100% oxygen for 64 h were studied at 0, 7 and 14 d after removal from the hyperoxic chamber and compared with control rats breathing room air. In the isolated-perfused, liquid-filled rat lung, the albumin flux from the perfusate into the air spaces increased immediately after the oxygen exposure (220 +/- 56 mg/h) and returned to control values (28 +/- 7 mg/h) after 7 and 14 d of recovery. The small solutes (Na+ and mannitol) flux across the alveolar epithelium normalized only after 14 d of recovery in room air. Active Na+ transport and lung liquid clearance were reduced by approximately 45% immediately after oxygen exposure when compared with control values, increased by approximately 56% above control values after 7 d of recovery, and returned to control values after 14 d of recovery. Paralleling these changes the Na,K-ATPase activity decreased by approximately 41% in AT2 cells isolated from rats after 64 h of breathing 100% O2 and increased by approximately 25% after the rats recovered in room air for 7 d. These results suggest that alveolar epithelial Na,K-ATPase may contribute in the recovery from the hyperoxic lung injury by participating in the clearance of lung edema.

Active Na+ transport and lung edema clearance were studied in a model of lung injury caused by sublethal oxygen exposure. Rats exposed to 85% O2 for 7 days were studied at 0, 7, 14, and 30 days after removal from the hyperoxic chamber and compared with room air controls. In the isolated-perfused, fluid-filled rat lung, albumin flux from the perfusate into the air spaces increased after oxygen exposure and returned to control values after 7 days of recovery. However, permeability to small solutes (Na+ and mannitol) normalized only after 30 days of recovery from hyperoxia. Active Na+ transport increased immediately after oxygen exposure and returned to control values 7 days after removal from hyperoxic chamber. Na-K-adenosinetriphosphatase (ATPase) activity, and protein expression in alveolar epithelial type II cells obtained at the end of the isolated lung experiments increased significantly after the oxygen exposure compared with controls in association with the increased active Na+ transport. We conclude that active Na+ transport and lung liquid clearance are increased in the subacute hyperoxic phase of lung injury in rats, due in part to the upregulation of alveolar epithelial Na-K-ATPases. Conceivably, this behavior protects against the effects of lung injury by allowing the injured lung to clear edema more effectively. Accordingly, this upregulation may be targeted as a strategy to diminish edema in patients with lung injury.

The isolated perfused liquid-filled rat lung in a "pleural bath" was the model used to study liquid exchange across the lung epithelium. Active transport and passive solute movement between the air space, the vascular perfusate, and the bath result in concentration changes of the three markers (Evans blue-tagged albumin, 22Na+, and [3H]mannitol) instilled in the air space. A mathematical model was developed to estimate the active and passive solute transports and to interpret the results. Rat lungs were perfused at left atrial and pulmonary arterial pressures of 0 and 8 mmHg, respectively. Six rat lung experiments were conducted at 37 degrees C and six at 4 degrees C. The normothermic experiments demonstrate that active transport accounts for 26% of the Na+ movement out of the air space (17.3 +/- 0.7 nm/s) and that passive mechanisms account for the remaining 74% (48.0 +/- 5.7 nm/s). Hypothermia inhibits lung liquid clearance but does not affect passive solute movement, suggesting that lung liquid clearance is effected by active Na+ transport mechanisms.

Pulmonary edema clearance is driven primarily by active sodium transport out of the alveoli, mediated predominantly by apical sodium channels and the basolateral NA,K-ATPase. We postulated that dopamine, analogous to its effects in other transporting epithelia, could regulate these sodium transport mechanisms and affect lung liquid clearance. We therefore studied the effects of dopamine on sodium transport and liquid clearance in isolated perfused rat lungs. Instillation of dopamine into the airways caused a dose-dependent increase in liquid clearance from isolated rat lungs of up to 33% above control values at 10(-8) to 10(-4) M concentrations. 10(-6) M amiloride, which selectively inhibits apical sodium channels, decreased basal liquid clearance by 34% but did not inhibit the dopamine-mediated stimulation of lung liquid clearance. Instillation of 10(-4) M amiloride into rat airways, which inhibits other sodium transport mechanisms non-selectively, decreased basal lung liquid clearance by 49% and inhibited the dopamine-mediated stimulation of lung liquid clearance. Perfusion of rat lungs with 5 x 10(-4) M ouabain to specifically inhibit Na,K-ATPase reduced both basal clearance (by 55%) and the dopamine-stimulated increase in lung fluid clearance. Conceivably, the stimulation of lung liquid clearance by dopamine is due to a modulation of Na,K-ATPase in the pulmonary epithelium.