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.
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.S hortly before birth, the fetal lung converts from fluid secretion to fluid reabsorption. After birth, efficient gas exchange depends on regulation of the amount of fluid in the thin (average, 0.2 m) liquid layer lining the alveolar epithelium (1). Alveolar flooding resulting from cardiogenic pulmonary edema or acute lung injury impairs gas diffusion across the air͞blood barrier; an increase in alveolar fluid clearance restores a normal air͞blood barrier. Alveolar fluid transport from alveolar to interstitial spaces, driven by active Na ϩ transport across the alveolar epithelium (2), can be inhibited either by the addition of amiloride, a Na ϩ channel inhibitor, to the alveolar space, or ouabain, a Na ϩ ,K ϩ -ATPase inhibitor, to the vascular bed (3), suggesting that the alveolar epithelium is the major site of Na ϩ transport and fluid absorption in the adult lung.The alveolar epithelium, which covers Ͼ99% of the large internal surface area of the lung (4), is composed of two cell types, alveolar type I (TI) and type II (TII) cells. TII cells, which cover 2-5% of the internal surface area of the lung, are cuboidal cells that synthesize and secrete pulmonary surfactant. TII cells contain ion channels, including the amiloride-sensitive epithelial Na ϩ channel (ENaC) (5), Na ϩ ,K ϩ -ATPase (3) and the cystic fibrosis transmembrane regulator (CFTR) (6). TI cells are large squamous cells whose thin cytoplasmic extensions cover Ͼ95% of the internal surface area of the lung (7). TI cells express aquaporin 5, a water channel (8), and have the highest known osmotic water permeability of any mammalian cell type (9). The observations that TII cells contain ion channels and TI cells express aquaporins led to the paradigm that TII cells govern alveolar fluid balance by regulating Na ϩ transport in the lungs, whereas TI cells merely provide a route for passive water absorpti...
Adenosine is a purine nucleoside that regulates cell function through G protein-coupled receptors that activate or inhibit adenylyl cyclase. Based on the understanding that cAMP regulates alveolar epithelial active Na ؉ transport, we hypothesized that adenosine and its receptors have the potential to regulate alveolar ion transport and airspace fluid content. Herein, we report that type 1 (A1R), 2a (A2aR), 2b (A2bR), and 3 (A3R) adenosine receptors are present in rat and mouse lungs and alveolar type 1 and 2 epithelial cells (AT1 and AT2). Rat AT2 cells generated and produced cAMP in response to adenosine, and micromolar concentrations of adenosine were measured in bronchoalveolar lavage fluid from mice. Ussing chamber studies of rat AT2 cells indicated that adenosine affects ion transport through engagement of A1R, A2aR, and/or A3R through a mechanism that increases CFTR and amiloride-sensitive channel function. Intratracheal instillation of low concentrations of adenosine (<10 ؊8 M) or either A2aR-or A3R-specific agonists increased alveolar fluid clearance (AFC), whereas physiologic concentrations of adenosine (>10 ؊6 M) reduced AFC in mice and rats via an A1R-dependent pathway. Instillation of a CFTR inhibitor (CFTRinh-172) attenuated adenosine-mediated down-regulation of AFC, suggesting that adenosine causes Cl ؊ efflux by means of CFTR. These studies report a role for adenosine in regulation of alveolar ion transport and fluid clearance. These findings suggest that physiologic concentrations of adenosine allow the alveolar epithelium to counterbalance active Na ؉ absorption with Cl ؊ efflux through engagement of the A1R and raise the possibility that adenosine receptor ligands can be used to treat pulmonary edema.active sodium transport ͉ adenosine receptors ͉ cystic fibrosis transmembrane conductance regulator P ulmonary edema is due to increased fluid flux into the airspace and impairment of the active Na ϩ transport that clears it (1-4). A variety of approaches to improve alveolar epithelial cell active Na ϩ transport for purposes of accelerating alveolar fluid clearance (AFC) have been explored in experimental systems. Of particular interest are receptor-ligand interactions that increase cAMP production in alveolar epithelial cells. Adenosine is a purine nucleoside that signals through four distinct G protein-coupled receptors, type 1 (A 1 R), type 2a (A 2a R), type 2b (A 2b R), and type 3 (A 3 R). In most cell systems, the A 1 R and A 3 R receptors inhibit adenylyl cyclase and/or lead to signaling through inositol-3-phosphate and phospholipase C. Engagement of type 2 receptors activates adenylyl cyclase by means of Gs␣ and increases cAMP levels. The ability of adenosine receptors (ARs) to couple to adenylyl cyclase led us to hypothesize that ARs might participate in regulation of alveolar epithelial active Na ϩ transport. We approached this hypothesis in rats and mice by testing whether adenosine and its receptors are present in the distal airspace and whether they affect AFC in vivo and vectorial Na ϩ...
Keratinocyte growth factor (KGF) has efficacy in several experimental models of lung injury; however, the mechanisms underlying KGF's protective effect remain incompletely understood. This study was undertaken to determine whether KGF augments barrier function in primary rat alveolar epithelial cells grown in culture, specifically whether KGF alters tight junction function via claudin expression. KGF significantly increased alveolar epithelial barrier function in culture as assessed by transepithelial electrical resistance (TER) and paracellular permeability. Fluorescence-activated cell sorting of freshly isolated type 1 (AT1) and type 2 (AT2) cells followed by quantitative real-time RT-PCR revealed that more than 97% of claudin mRNA transcripts in these cells were for claudins-3, -4, and -18. Using cultured AT2 cells, we then examined the effect of KGF on the protein levels of the claudins with the highest mRNA levels: -3, -4, -5, -7, -12, -15, and -18. KGF did not alter the levels of any of the claudins tested, nor of zona occludens-1 (ZO-1) or occludin. Moreover, localization of claudins-3, -4, -18, and ZO-1 was unchanged. KGF did induce a marked increase in the apical perijunctional F-actin ring. Actin depolymerization with cytochalasin D blocked the KGF-mediated increase in TER without significantly changing TER in control cells. Together, these data support a novel mechanism by which KGF enhances alveolar barrier function, modulation of the actin cytoskeleton. In addition, these data demonstrate the complete claudin expression profile for AT1 and AT2 cells and indicate that claudins-3, -4, and -18 are the primary claudins expressed in these cell types.
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