Mechanisms of alveolar epithelial chloride absorption

Circulation Research, 2003

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. (Circ Res. 2003;92: 453-460.)

Proceedings of the National Academy of Sciences, 2002

Transport of lung liquid is essential for both normal pulmonary physiologic processes and for resolution of pathologic processes. The large internal surface area of the lung is lined by alveolar epithelial type I (TI) and type II (TII) cells; TI cells line >95% of this surface, TII cells <5%. Fluid transport is regulated by ion transport, with water movement following passively. Current concepts are that TII cells are the main sites of ion transport in the lung. TI cells have been thought to provide only passive barrier, rather than active, functions. Because TI cells line most of the internal surface area of the lung, we hypothesized that TI cells could be important in the regulation of lung liquid homeostasis. We measured both Na ؉ and K ؉ (Rb ؉ ) transport in TI cells isolated from adult rat lungs and compared the results to those of concomitant experiments with isolated TII cells. TI cells take up Na ؉ in an amiloride-inhibitable fashion, suggesting the presence of Na ؉ channels; TI cell Na ؉ uptake, per microgram of protein, is Ϸ2.5 times that of TII cells. Rb ؉ uptake in TI cells was Ϸ3 times that in TII cells and was inhibited by 10 ؊4 M ouabain, the latter observation suggesting that TI cells exhibit Na ؉ -, K ؉ -ATPase activity. By immunocytochemical methods, TI cells contain all three subunits (␣, ␤, and ␥) of the epithelial sodium channel ENaC and two subunits of Na ؉ -, K ؉ -ATPase. By Western blot analysis, TI cells contain Ϸ3 times the amount of ␣ENaC͞g protein of TII cells. Taken together, these studies demonstrate that TI cells not only contain molecular machinery necessary for active ion transport, but also transport ions. These results modify some basic concepts about lung liquid transport, suggesting that TI cells may contribute significantly in maintaining alveolar fluid balance and in resolving airspace edema.

American journal of physiology. Lung cellular and molecular physiology, 2002

Despite a presumptive role for type I (AT1) cells in alveolar epithelial transport, specific Na transporters have not previously been localized to these cells. To evaluate expression of Na transporters in AT1 cells, double labeling immunofluorescence microscopy was utilized in whole lung and in cytocentrifuged preparations of partially purified alveolar epithelial cells (AEC). Expression of Na pump subunit isoforms and the alpha-subunit of the rat (r) epithelial Na channel (alpha-ENaC) was evaluated in isolated AT1 cells identified by their immunoreactivity with AT1 cell-specific antibody markers (VIIIB2 and/or anti-aquaporin-5) and lack of reactivity with antibodies specific for AT2 cells (anti-surfactant protein A) or leukocytes (anti-leukocyte common antigen). Expression of the Na pump alpha(1)-subunit in AEC was assessed in situ. Na pump subunit isoform and alpha-rENaC expression was also evaluated by RT-PCR in highly purified (approximately 95%) AT1 cell preparations. Labeling ...

American Journal of Physiology-Lung …, 2003

lyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na ϩ transport and, in particular, the role of amiloride-sensitive Na ϩ channels in the apical membrane and the Na ϩ-K ϩ-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl Ϫ and K ϩ channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl Ϫ and K ϩ channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.

American journal of respiratory cell and molecular biology, 2016

Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and relative contributions of alveolar epithelial type II (AT2) vs type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which β1 subunit was knocked out in either AT1 cells or in both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing for the first time that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung wei...

Journal of Tissue Culture Methods, 1992

To investigate the celt physiologic and biological properties of the alveolar epithelium, we studied rat alveolar epithelial cell monolayers grown on permeable supports in primary culture. Type II alveolar epithelial cells were disaggregated using elastase, and partially purified on a discontinuous metrizamide gradient. These isolated cells were plated onto tissue culture-treated Nuclepore membrane filters at 1.5 )< 106 cells/cm 2 and maintained in a humidified incubator (5% CO2 in air, 37 ° C). After 2 days in culture, the bathing media on both sides of the cell monolayers were changed to fresh culture medium, thus removing nonadherent cells (mostly lcukocytes). These monolayers exhibit a high transmonolayer resistance (>2000 ~-cm 2) and actively transport ions. Radionuclide flux studies indicate that Na + is the predominant ionic species absorbed actively under baseline conditions, accounting for about 80% of the total active ion transport. CI-seems to be passively transported across the epithelium. However, when the epithelium is exposed to a beta-agonist (terbutaline), active absorption of Na + is increased ar, d active absorption of C1-occurs. Ahhough it is clear that both active Na + and CI-transport are dependent on Na+/K+-ATPase activity, and that Na + enters cells predominantly through channels, the specific mechanisms by which CI-enters and exits the alveolar epithelial cells remain unclear. The stimulated reabsorption of Na + and CImay be important in helping to remove excess fluid from alveolar air spaces in the lung.

Circulation Research, 2004

Alveolar epithelial beta-adrenergic receptor (betaAR) activation accelerates active Na+ 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 beta1- or beta2-adrenergic receptors (beta1AR-/-/beta2AR-/-) 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 betaAR signaling may not be required for alveolar fluid homeostasis in uninjured lungs. Comparison of isoproterenol-sensitive AFC in mice with beta1- but not beta2-adrenergic receptors to beta1AR-/-/beta2AR-/- mice indicates that the beta2AR mediates the bulk of beta-adrenergic-sensitive alveolar active Na+ transport. To test the necessity of betaAR signaling in acute lung injury, beta1AR-/-/beta2AR-/-, beta1AR+/+/beta2AR-/-, and beta1AR+/+/beta2AR+/+ mice were exposed to 100% oxygen for up to 204 hours. beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice had more lung water and worse survival from this form of acute lung injury than wild-type controls. Adenoviral-mediated rescue of beta2-adrenergic receptor (beta2AR) function into the alveolar epithelium of beta1AR-/-/beta2AR-/- and beta1AR+/+/beta2AR-/- mice normalized distal lung beta2AR function, alveolar epithelial active Na+ transport, and survival from hyperoxia. These findings indicate that betaAR signaling may not be necessary for basal AFC, and that beta2AR is essential for the adaptive physiological response needed to clear excess fluid from the alveolar airspace of normal and injured lungs.

American journal of physiology. Lung cellular and molecular physiology, 2000

In this review, we discuss evidence that supports the hypothesis that adrenergic stimulation of transepithelial Na absorption across the alveolar epithelium occurs indirectly by activation of apical Cl channels, resulting in hyperpolarization and an increased driving force for Na uptake through amiloride-sensitive Na channels. This hypothesis differs from the prevailing idea that adrenergic-receptor activation increases the open probability of Na channels, leading to an increase in apical membrane Na permeability and an increase in Na and fluid uptake from the alveolar space. We review results from cultured alveolar epithelial cell monolayer experiments that show increases in apical membrane Cl conductance in the absence of any change in Na conductance after stimulation by selective beta-adrenergic-receptor agonists. We also discuss possible reasons for differences in Na-channel regulation in cells grown in monolayer culture compared with that in dissociated alveolar epithelial cell...

Proceedings of the National Academy of Sciences, 2006

Efficient gas exchange in the lungs depends on regulation of the amount of fluid in the thin (average 0.2 m) liquid layer lining the alveolar epithelium. Fluid fluxes are regulated by ion transport across the alveolar epithelium, which is composed of alveolar type I (TI) and type II (TII) cells. The accepted paradigm has been that TII cells, which cover <5% of the internal surface area of the lung, transport Na ؉ and Cl ؊ and that TI cells, which cover >95% of the surface area, provide a route for water absorption. Here we present data that TI cells contain functional epithelial Na ؉ channels (ENaC), pimozide-sensitive cation channels, K ؉ channels, and the cystic fibrosis transmembrane regulator. TII cells contain ENaC and cystic fibrosis transmembrane regulator, but few pimozide-sensitive cation channels. These findings lead to a revised paradigm of ion and water transport in the lung in which (i) Na ؉ and Cl ؊ transport occurs across the entire alveolar epithelium (TI and TII cells) rather than only across TII cells; and (ii) by virtue of their very large surface area, TI cells are responsible for the bulk of transepithelial Na ؉ transport in the lung.

Respiratory Physiology & Neurobiology, 2004

Native alveolar epithelium from Xenopus lung was used for electrophysiological Ussing chamber experiments to investigate ion transport regulation. The tissue exhibits a considerable absorption of Na + ions and this transepithelial transport is largely up-regulated after treatment of donor animals with ACTH. Extracellular ATP, UTP and adenosine were tested for their regulating effects and all three increased I sc , which was mainly due to a stimulation of amiloride sensitive Na + transport (increase of I ami 32% for ATP, 21% for UTP, 25% for adenosine). Solely the effect of UTP was completely abolished in the presence of amiloride. In contrast, the effects of ATP or adenosine disappeared under Cl − -free conditions. ATP and UTP proved to have additive effects and pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid (PPADS), an antagonist of purinergic receptors, inhibited selectively the effect of UTP on I sc . Further, I sc was increased by the P2X selective agonist ␤,␥-meATP. We were able to demonstrate, that extracellular purines and pyrimidines play a possible role as auto/paracrine messengers for alveolar ion transport regulation in Xenopus lung.