Adult Alveolar Epithelial Cells Research Articles

Integrated Transcriptomic and Epigenomic Analysis of Primary Human Lung Epithelial Cell Differentiation

Elucidation of the epigenetic basis for cell-type specific gene regulation is key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how epigenetic changes are integrated with transcriptional activation to determine cell phenotype during differentiation. We performed epigenomic profiling in conjunction with transcriptomic profiling using in vitro differentiation of human primary alveolar epithelial cells (AEC). This model recapitulates an in vivo process in which AEC transition from one differentiated cell type to another during regeneration following lung injury. Interrogation of histone marks over time revealed enrichment of specific transcription factor binding motifs within regions of changing chromatin structure. Cross-referencing of these motifs with pathways showing transcriptional changes revealed known regulatory pathways of distal alveolar differentiation, such as the WNT and transforming growth factor beta (TGFB) pathways, and putative novel regulators of adult AEC differentiation including hepatocyte nuclear factor 4 alpha (HNF4A), and the retinoid X receptor (RXR) signaling pathways. Inhibition of the RXR pathway confirmed its functional relevance for alveolar differentiation. Our incorporation of epigenetic data allowed specific identification of transcription factors that are potential direct upstream regulators of the differentiation process, demonstrating the power of this approach. Integration of epigenomic data with transcriptomic profiling has broad application for the identification of regulatory pathways in other models of differentiation.

Chloride secretion across adult alveolar epithelial cells contributes to cardiogenic edema

In utero, fetal lung epithelial cells actively secrete chloride (Cl−) ions into the lung airspaces. Cl− ions enter the basolateral membranes through Na+/K+/2Cl− (NKCC) transporters (1), down an electrochemical gradient generated by the basolateral Na+/K+/ATPase, and exit through apical anion channels, including the cAMP- activated cystic fibrosis transmembrane regulator (CFTR) (2). Na+ ions follow passively through the paracellular junctions to preserve electroneutrality. The vectorial transport of NaCl generates an oncotic pressure, which drives fetal fluid secretion into the airway lumen. Shortly before birth, Cl− secretion diminishes and active Na+ transport begins: Na+ ions enter lung epithelial cells through amiloride-sensitive Na+ channels (ENaC) and are extruded across the basolateral membranes by the Na+/K+/ATPase (3, 4). This process is critical for the reabsorption of fetal lung fluid and the establishment of gas exchange. In the adult lung active Na+ reabsorption limits the degree of alveolar edema in hyperoxic lung injury (5) and in patients with acute respiratory distress syndrome (6, 7).

Integrin α6β4 identifies an adult distal lung epithelial population with regenerative potential in mice

Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6β4, but little or no pro-surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin β4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified β4+ adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10+) airway-like and SPC+ saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC- progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair.

ErbB4 REGULATES SURFACTANT SYNTHESIS AND PROLIFERATION IN ADULT RAT PULMONARY EPITHELIAL CELLS

ErbB4 is a predominant heterodimer for other ErbB receptors in late fetal lung development where it participates in regulating type II cell surfactant synthesis. To further elucidate the role of ErbB4 in pulmonary alveolar epithelial cell function, the authors hypothesized that ErbB4 participates in maintaining adult lung type II cell homeostasis. The authors used small interfering RNA (siRNA) to down-regulate endogenous, ErbB4 receptors in the adult rat lung epithelial L2 cell line and measured neuregulin 1β (NRG1β)-, and fibroblast conditioned medium (FCM)-induced effects on L2 cell surfactant phospholipid synthesis and proliferation. Under control conditions, total and phosphorylated ErbB4 were significantly increased after both NRG1β and FCM treatment, as were surfactant phospholipids synthesis and cell proliferation. Down-regulation of ErbB4 with siRNA reduced stimulation of NRG1β- and FCM-induced ErbB4 phosphorylation, decreased endogenous surfactant phospholipid synthesis, and blocked NRG1β- and FCM-stimulated surfactant phospholipid synthesis. NRG1β- and FCM-induced cell proliferation was not affected. The authors conclude that ErbB4 participates in maintaining adult lung alveolar epithelial cell surfactant synthesis and proliferation with development-specific functions.

Cell-specific signal transduction pathways regulating Na+-K+-ATPase. Focus on “Short-term effects of thyroid hormones on the Na+-K+-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction”

the na+-k+-atpase is a complex integral membrane protein that carries out active transport of sodium and potassium across the cell plasma membrane, maintaining the ionic gradients. Each subunit has multiple isozymes that are expressed in tissue and developmental specific fashion. In addition to

T3 increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells

Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and beta-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase alpha(1)-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.

Vascular endothelial growth factor promotes physical wound repair and is anti-apoptotic in primary distal lung epithelial and A549 cells

There is evidence to suggest a beneficial role for growth factors, including vascular endothelial growth factor (VEGF), in tissue repair and proliferation after injury within the lung. Whether this effect is mediated predominantly by actions on endothelial cells or epithelial cells is unknown. This study tested the hypothesis that VEGF acts as an autocrine trophic factor for human adult alveolar epithelial cells and that under situations of pro-apoptotic stress, VEGF reduces cell death. In vitro cell culture study looking at the effects of 0.03% H2O2 on both A549 and primary distal lung epithelial cells. Primary adult human distal lung epithelial cells express both the soluble and membrane-associated VEGF isoforms and VEGF receptors 1 and 2. At physiologically relevant doses, soluble VEGF isoforms stimulate wound repair and have a proliferative action. Specific receptor ligands confirmed that this effect was mediated by VEGF receptor 1. In addition to proliferation, we demonstrate that VEGF reduces A549 and distal lung epithelial cell apoptosis when administered after 0.03% H2O2 injury. This effect occurs due to reduced caspase-3 activation and is phosphatidylinositol 3'-kinase dependent. In addition to its known effects on endothelial cells, VEGF acts as a growth and anti-apoptotic factor on alveolar epithelial cells. VEGF treatment may have potential as a rescue therapy for diseases associated with alveolar epithelial damage such as acute respiratory distress syndrome.

The role of cystic fibrosis transmembrane conductance regulator chloride channel in β-receptor-mediated regulation of alveolar fluid clearance

LETTERS TO THE EDITORThe role of cystic fibrosis transmembrane conductance regulator chloride channel in β-receptor-mediated regulation of alveolar fluid clearanceMichael EisenhutMichael EisenhutPublished Online:01 Jul 2007https://doi.org/10.1152/japplphysiol.00280.2007MoreSectionsPDF (30 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat to the editor: A recent study (6) reported on an association of genetically determined reduced β-receptor function with increased pulmonary fluid accumulation after fluid challenge with rapid normal saline infusion in healthy human volunteers. The authors emphasized that key pathways by which β-receptors can prevent excessive accumulation of fluid in the lungs increase the number and activity of epithelial sodium channels (ENaC) and Na-K-ATPase. A recent study on changes in epithelial ion transport in children with septicemia-induced pulmonary edema revealed that severity of respiratory compromise (ventilation index) and duration of ventilation were more closely related to parameters reflecting systemic epithelial chloride rather than sodium transport in airways, sweat, and salivary gland (1). The authors of the present study referred to a study demonstrating increased airway liquid in patients with congenital ENaC inactivation (pseudohypoaldosteronism). This study however did not demonstrate increased alveolar fluid in these patients. A previous study challenging mice homozygous for a mutation inactivating the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with a fluid overload with normal saline found an increase in alveolar liquid (3). CFTR has recently been found to contribute independently to fluid transport in alveolar epithelial type II cells in vitro (4). CFTR is activated by protein kinase A in a cAMP- and, therefore, β-receptor-dependent way (2). CFTR activation by cAMP in alveolar epithelial cells has been shown to be a condition for apical sodium influx into alveolar epithelial cells through ENaC by hyperpolarization (5). The role of CFTR in β-receptor-dependent alveolar fluid clearance may be equally or more important than the role of ENaC or Na-K-ATPase. Future studies could compare the response of airway epithelial sodium and chloride channels to β-agonists in vivo as demonstrated previously (1) as an explanatory outcome to assess the effectiveness of β-agonists in stimulation of chloride transport in patients with different β-receptor genotype groups not only affecting receptor density but also receptor function.REFERENCES1 Eisenhut M, Wallace H, Barton P, Gaillard E, Newland P, Diver M, Southern KW. Pulmonary edema in meningococcal septicemia associated with reduced epithelial chloride transport. Ped Crit Care Med 7: 119–124, 2006.Crossref | PubMed | ISI | Google Scholar2 Eisenhut M. Changes in ion transport in inflammatory disease. J Inflammation (Lond) 3: 1–15, 2006.Crossref | Google Scholar3 Fang X, Fukuda N, Barbry P, Sartori C, Verkman AS, Matthay MA. Novel role of CFTR in fluid absorption from the distal airspaces of the lung. J Gen Physiol 119: 199–207, 2002.Crossref | PubMed | ISI | Google Scholar4 Fang X, Song Y, Hirsch J, Galietta LJ, Pedemonte N, Zemans RL, Dolganov G, Verkman AS, Matthay MA. Contribution of CFTR to apical-basolateral fluid transport in cultured human alveolar epithelial type II cells. Am J Physiol Lung Cell Mol Physiol 290: 242–249, 2006.Link | ISI | Google Scholar5 O'Grady SM, Jiang X, Ingbar DH. Cl-channel activation is necessary for stimulation of Na transport in adult alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 278: L239–L244, 2000.Link | ISI | Google Scholar6 Snyder EM, Beck KC, Turner ST, Hoffman EA, Joyner MJ, Johnson BD. Genetic variation of the β2-adrenergic receptor is associated with differences in lung fluid accumulation in humans. J Appl Physiol 102: 2172–2178, 2007.Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: M. Eisenhut, Luton & Dunstable Hospital NHS Foundation Trust, Lewsey Road, Luton, Bedfordshire, UK (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationCited ByAmiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl − conductance16 January 2014 | Physiological Reports, Vol. 2, No. 1Reply to EisenhutEric M. Snyder, Stephen T. Turner, and Bruce D. Johnson1 July 2007 | Journal of Applied Physiology, Vol. 103, No. 1 More from this issue > Volume 103Issue 1July 2007Pages 413-413 Copyright & PermissionsCopyright © 2007 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00280.2007PubMed17615287History Published online 1 July 2007 Published in print 1 July 2007 Metrics

Developmental acquisition of T3-sensitive Na-K-ATPase stimulation by rat alveolar epithelial cells

Late in gestation, the developing air space epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na-K-ATPase acting in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption, and thyroid hormone [triiodothyronine (T3)] stimulates adult alveolar epithelial cell (AEC) Na-K-ATPase. This study explored the developmental regulation of Na-K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) cells. T3 increased Na-K-ATPase activity in primary FDLE cells from gestational day 19 [both primary FDLE cells at embryonic day 19 (E19) and the cell line FD19 derived from FDLE cells at E19]. However, T3 did not increase the Na-K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the cell line FD18 (derived from FDLE cells at E18). Subsequent experiments assessed the T3 signal pathway to define whether it was similar in the late FDLE and adult AEC and to determine the site of the switch in responsiveness to T3. As in adult AEC, in the FD19 cell line, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked the T3-induced increase in Na-K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active mutant of the PI3K catalytic p110 subunit significantly augmented the Na-K-ATPase activity and the cell surface expression of Na-K-ATPase alpha(1) protein. In conclusion, FDLE cells from E17 and E18 lacked T3-sensitive Na-K-ATPase activity but acquired this response at E19. The developmental stimulation of Na-K-ATPase by T3 in rat FDLE cells requires activation of PI3K, and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway.

Thyroid hormone and Na+-K+-ATPase: more than simple transcription

FLUID SECRETION INTO THE DEVELOPING lungs’ air spaces is essential for normal lung development, but this fluid must be cleared at birth so that effective gas exchange can occur. Impaired clearance of this fetal lung liquid, which arises from immaturity of the distal lung epithelium’s active Na transport system (for review, see Ref. 17), causes transient tachypnea of the newborn (2, 12, 13), and, if combined with immaturity of the surfactant system, neonatal respiratory distress syndrome (nRDS) (4, 17). Clearance of air space fluid is driven by active transepithelial Na transport resulting from synchronized activity of apical Na permeant ion channels (e.g., epithelial Na channel, ENaC) and basolateral Na-K-ATPase. The chemical and electrical gradients for Na absorption are, respectively, maintained by Na-K-ATPase and K channels. Perinatal changes in PO2, glucocorticoids, -agonists, and thyroid hormones interact to increase transepithelial Na absorption, to bring about the switch from net fluid secretion to net absorption as lung liquid is cleared at birth. However, despite considerable data from molecular, transgenic, cell culture, and in vivo studies, the fundamental mechanisms of this crucial developmental shift are incompletely understood (5, 19). Barker and coworkers (6) demonstrated that thyroid hormone [triiodothyronine (T3)] played an important role in maturation of the fetal lungs’ ability to respond to an infusion of epinephrine by switching to fluid absorption, and others showed that thyroid hormone in concert with glucocorticoids increased the expression of ENaC (18, 22). Although T3 classically acts as a direct transcriptional activator of target genes and has been shown to potentiate dexamethasone-stimulated transcription of the ENaC promoter (20), T3-induced upregulation of Na-K-ATPase activity in adult rat alveolar epithelial cells was recently shown to be insensitive to actinomycin D (15). This has functional significance. For example, it has been demonstrated that overexpression of Na-KATPase via adenoviral-mediated gene transfer is sufficient to increase alveolar fluid clearance (21). It is known that thyroid hormones have profound effects on the maturation of the fetal lung and that premature infants with nRDS have lower thyroid hormone levels than well preterm infants (9, 10). However, when clinical trials have been conducted wherein augmentation of the thyroid hormonal axis has taken place, there has been no demonstrable benefit. The THORN trial of T3 and hydrocortisone supplementation in preterm infants of less than 30 wk gestation found that low thyroid hormone levels were associated with higher mortality and ventilator dependence (7, 8). However, this trial (8) was unable to demonstrate beneficial effects of T3 and hydrocortisone infusion. Similarly, the provision of thyroid-releasing hormone to infants who had received antenatal glucocorticoid

Dexamethasone responsive element in the rat Na, K-ATPase β 1 gene coding region

The Na, K-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase Na, K-ATPase activity, mRNA levels, and transcription rate of the β 1 subunit. In this study, we describe a glucocorticoid responsive element (GRE) in the coding region of the rat Na, K-ATPase β 1 gene in a rat lung epithelial cell line. Transient transfection experiments with the β 1 subunit coding region with or without the 5′ and 3′ untranslated regions demonstrated responsiveness to dexamethasone induction and also identified a GRE at +434 in exon IV. The +434 GRE conferred dexamethasone responsiveness in a heterologous thymidine kinase promoter irrespective of its orientation to the β 1 promoter. Transcriptional upregulation by dexamethasone was abolished in +434 mutants. Electrophoretic mobility shift assays (EMSA) demonstrated specific binding of nuclear proteins to the +434 GRE and the presence of the GC receptor. This specific binding was inhibited by a GRE previously described in the rat Na, K-ATPase β 1 gene at −631. In conclusion, we identified a GRE at +434 in the exon IV of the rat Na, K-ATPase β 1 gene.

Dexamethasone stimulates transcription of the Na+-K+-ATPase beta1 gene in adult rat lung epithelial cells.

Na+-K+-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase Na+-K+-ATPase activity and mRNA levels. We sought to define the mechanism of Na+-K+-ATPase gene upregulation by GC. In a rat alveolar epithelial cell line (RLE), dexamethasone (Dex) increased beta1-subunit Na+-K+-ATPase mRNA expression two- to threefold within 3 h after exposure to the GC. The increased gene expression was due to increased transcription as demonstrated by nuclear run-on assays, whereas mRNA stability remained unchanged. Transient transfection of 5' deletion mutants of a beta1 promoter-reporter construct demonstrated a 1.5- to 2.2-fold increase in promoter activity by Dex. All of the 5' deletion constructs contained partial or palindromic GC regulatory elements (GRE) and responded to GC. The increased expression of promoter reporter was inhibited by RU-486, a GC receptor (GR) antagonist, suggesting the involvement of GR. The palindromic GRE at -631 demonstrated Dex induction in a heterologous promoter construct. Gel mobility shift assays using RLE nuclear extracts demonstrated specific binding to this site and the presence of GR. We conclude that GC directly stimulate transcription of Na+-K+-ATPase beta1 gene expression in adult rat lung epithelial cells through a GR-dependent mechanism that can act at multiple sites.

Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane.

Whole cell perforated patch-clamp experiments were performed with adult rat alveolar epithelial cells. The holding potential was -60 mV, and depolarizing voltage steps activated voltage-gated K(+) (Kv) channels. The voltage-activated currents exhibited a mean reversal potential of -32 mV. Complete activation was achieved at -10 mV. The currents exhibited slow inactivation, with significant variability in the time course between cells. Tail current analysis revealed cell-to-cell variability in K(+) selectivity, suggesting contributions of multiple Kv alpha-subunits to the whole cell current. The Kv channels also displayed steady-state inactivation when the membrane potential was held at depolarized voltages with a window current between -30 and 5 mV. Analysis of RNA isolated from these cells by RT-PCR revealed the presence of eight Kv alpha-subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1, Kv4.2, Kv4.3, and Kv9.3), three beta-subunits (Kvbeta1.1, Kvbeta2.1, and Kvbeta3.1), and two K(+) channel interacting protein (KChIP) isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv alpha-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showed labeling of 50-kDa proteins from alveolar epithelial cells grown in monolayer culture. Immunocytochemical analysis of cells from monolayers showed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to the apical membrane. We conclude that expression of multiple Kv alpha-, beta-, and KChIP subunits explains the variability in inactivation gating and K(+) selectivity observed between cells and that Kv channels in the apical membrane may contribute to basal K(+) secretion across the alveolar epithelium.

Modification of sodium transport and alveolar fluid clearance by hypoxia: mechanisms and physiological implications.

In order for gas exchange to occur optimally, the alveoli must remain open and free from fluid. In utero, the fetal lung is filled with fluid which is removed shortly after birth, mainly because active reabsorption of sodium ions (Na ) across the alveolar epithelium creates an osmotic force favoring reabsorption of alveolar fluid (1, 2). The classic studies from Dr. Matthay’s laboratory showing reabsorption of intratracheally instilled isotonic fluid or plasma from the alveolar spaces of adult anesthetized animals, and the partial inhibition of this process by amiloride and ouabain, implied that adult alveolar epithelial cells are also capable of actively transporting sodium (Na ) ions (reviewed in Ref. 3). In the adult lung, active Na reabsorption plays an important role in limiting the degree of alveolar edema in pathologic conditions in which the alveolar epithelium has been damaged. For example, blocking Na transport increased lung water in rats exposed to hyperoxia (4). Conversely, intratracheal instillation of adenoviral vectors containing copies of the Na ,K -ATPase genes increased survival of rats exposed to hyperoxia (5). Patients with acute lung injury who were able to concentrate alveolar protein (as a result of active Na reabsorption) had a better prognosis than those that did not (6, 7). Presently, it has not been definitely established whether or not active Na transport also plays an important role in maintaining the normal alveoli free of fluid. Additional insight into the nature and regulation of transport pathways has been derived from electrophysiologic studies in freshly isolated and cultured alveolar type II (ATII) cells: Na ions diffuse passively into ATII cells through apically located amiloride-sensitive, amilorideinsensitive, and cGMP-gated cation channels with conductances of 4–25 pS (8, 9) and are extruded across the basolateral membranes by the ouabain-sensitive Na ,K -ATPase (10). To preserve neutrality, chloride (Cl ) ions move from the apical to the basolateral compartments either through the paracellular junctions and/or through chloride channels located in alveolar epithelial cells (11, 12). In situ hybridization studies identified the presence of two of the three subunits of the cloned epithelial Na channel ( ENaC and ENaC) in the alveolar region of both fetal and adult lungs (13). Currently there is controversy as to whether ENaC per se or ENaC-type channels (i.e., channels with biophysical properties distinct from those of ENaC) are the main pathways for Na entry into ATII cells (14). There have been numerous studies attempting to identify whether decreased alveolar fluid clearance (AFC) contributes to alveolar edema formation in a variety of pathophysiologic conditions. The results of several studies suggest that severe alveolar hypoxia results in decreased AFC and Na transport ( see Table 1). This is of major interest because alveolar hypoxemia may be encountered in a variety of pathologic conditions including hypoventilation, obstructive lung disease, and ascent to high altitude (15). To investigate the mechanisms responsible for the downregulation of AFC during hypoxia, Vivona and colleagues exposed rats to physiologic levels of hypoxia (8% O 2 for up to 24 h) and measured AFC in situ and ENaC and Na ,K -ATPase mRNA and protein in isolated ATII cells (16). They reported that alveolar hypoxia decreased both the total and amiloride-sensitive portion of AFC and that these changes were ameliorated by intratracheal instillation of a 2 agonist. Surprisingly, levels of -ENaC and 1 and 1 -Na ,K -ATPase in ATII cells remained unchanged. These findings are highly significant because not only do they provide new insight into the mechanism of edema formation during ascent to high altitude, but they also highlight a potential therapeutic strategy to decrease edema and improve gas exchange. Furthermore, the results of these studies clearly point out that it is not always possible to extrapolate changes in function from biochemical and molecular biology measurements. The methodology of measuring AFC in vivo was introduced by Matthay and coworkers almost twenty years ago (17). In this set of experiments, AFC was measured in nonventilated rats with cardiac arrest. At first glance, this seems contradictory to the basic premise, namely that alveolar fluid clearance depends on the presence of an energy-requiring Na ,K -ATPase. However, previous studies have shown that constant levels of AFC can be maintained for brief periods of time following cessation of ventilation ( Received in original form August 23, 2001 )

Adrenergic regulation of ion transport across adult alveolar epithelial cells: effects on Cl- channel activation and transport function in cultures with an apical air interface.

The effect of beta-adrenergic receptor stimulation on Cl- channel activation was investigated in alveolar epithelial cells grown in monolayer culture and in freshly isolated cells. Monolayers cultured under apical air interface conditions exhibited enhanced amiloride-sensitive Na+ transport compared to apical liquid interface monolayers. Amiloride or benzamil inhibited most (66%) of the basal short circuit current (Isc) with half-maximal inhibitory concentration (IC50) values of 0.62 microm and 0.09 microm respectively. Basolateral addition of terbutaline (2 microm) produced a rapid decrease in Isc followed by a slow recovery that exceeded the basal Isc. When Cl- was replaced with methanesulfonate in either intact monolayers or basolateral membrane permeabilized monolayers, the response to terbutaline (2 microm) was completely inhibited. No effect of terbutaline on amiloride-sensitive Na+ current was detected. beta-Adrenergic agonists and 8-chlorothiophenyl cyclic adenosine monophosphate (8-ctp cAMP) directly stimulated a Cl- channel in freshly isolated alveolar epithelial cells. The current was blocked by glibenclamide (100 microm) and had a reversal potential of -22 mV. No increase in amiloride-sensitve current was detected in response to terbutaline or 8-cpt cAMP stimulation. These data support the conclusion that beta-adrenergic agonists produce acute activation of apical Cl- channels and that monolayers maintained under apical air interface conditions exhibit increased Na+ absorption.

Selectivity properties of a Na-dependent amino acid cotransport system in adult alveolar epithelial cells.

We investigated the amino acid specificity of a Na-dependent amino acid cotransport system that contributes to transepithelial Na absorption in the apical membrane of cultured adult rat alveolar epithelial cell monolayers. Short-circuit current was increased by basic, uncharged polar, and nonpolar amino acids but not by L-aspartic acid or L-proline. EC(50) values for L-lysine and L-histidine were 0.16 and 0.058 mM, respectively. The L-lysine-stimulated short-circuit current was Na dependent, with a concentration causing a half-maximal stimulation by Na of 44.24 mM. L-Serine, L-glutamine, and L-cysteine had EC(50) values of 0.095, 0.25, and 0.12 mM, respectively. L-Alanine had the highest affinity, with an EC(50) of 0.027 mM. We conclude that monolayer cultures of adult rat alveolar epithelial cells possess a broad-specificity Na-dependent amino acid cotransport system with properties consistent with system B(0,+). We suggest that this cotransport system plays a critical role in recycling of constituent amino acids that make up glutathione, thus ensuring efficient replenishment of this important antioxidant within the alveolar fluid.

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.

A Cysteine Residue in the Helix-Loop-Helix Domain of Id2 Is Critical for Homodimerization and Function

Id proteins are negative regulators of basic helix-loop-helix (bHLH) transcription factors. In this study, we compared the expression of Id2 mRNA in proliferating (fetal) and nonproliferating (adult) alveolar epithelial cells (AECs). The expression of Id2 was higher in adult AECs than in the corresponding fetal cells, suggesting that Id2 might play a functional role in developmental regulation of lung epithelial cell proliferation. By screening a mouse embryo cDNA library in the yeast two-hybrid system, Id2 was identified as a self-associating protein. Structural analysis by deletion and site-directed mutagenesis demonstrated that the HLH domain and a cysteine residue within the HLH domain are essential for Id2 homodimerization. Furthermore, in vitro synthesized Id2 homodimers became monomers under reducing conditions, indicating that the formation of an intermolecular disulfide bond is critical for Id2 homodimerization. Transient transfection assays in A549 cells showed that wild-type Id2 down-regulated the activity of the cyclin A promoter by 70%, while mutating the cysteine critical for Id2 homodimerization abolished the inhibitory effect of wild-type Id2.

Cl-channel activation is necessary for stimulation of Na transport in adult alveolar epithelial cells.

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 cells. Finally, we describe some preliminary in vivo data that suggest a role for Cl-channel activation in the process of amiloride-sensitive alveolar fluid absorption.

Mechanical force-induced signal transduction in lung cells.

The lung is a unique organ in that it is exposed to physical forces derived from breathing, blood flow, and surface tension throughout life. Over the past decade, significant progress has been made at the cellular and molecular levels regarding the mechanisms by which physical forces affect lung morphogenesis, function, and metabolism. With the use of newly developed devices, mechanical forces have been applied to a variety of lung cells including fetal lung cells, adult alveolar epithelial cells, fibroblasts, airway epithelial and smooth muscle cells, pulmonary endothelial and smooth muscle cells, and mesothelial cells. These studies have led to new insights into how cells sense mechanical stimulation, transmit signals intra- and intercellularly, and regulate gene expression at the transcriptional and posttranscriptional levels. These advances have significantly increased our understanding of the process of mechanotransduction in lung cells. Further investigation in this exciting research field will facilitate our understanding of pulmonary physiology and pathophysiology at the cellular and molecular levels.