Fetal Distal Lung Epithelial Cells Research Articles

Mechanical properties of the premature lung: From tissue deformation under load to mechanosensitivity of alveolar cells.

Many preterm infants require mechanical ventilation as life-saving therapy. However, ventilation-induced overpressure can result in lung diseases. Considering the lung as a viscoelastic material, positive pressure inside the lung results in increased hydrostatic pressure and tissue compression. To elucidate the effect of positive pressure on lung tissue mechanics and cell behavior, we mimic the effect of overpressure by employing an uniaxial load onto fetal and adult rat lungs with different deformation rates. Additionally, tissue expansion during tidal breathing due to a negative intrathoracic pressure was addressed by uniaxial tension. We found a hyperelastic deformation behavior of fetal tissues under compression and tension with a remarkable strain stiffening. In contrast, adult lungs exhibited a similar response only during compression. Young’s moduli were always larger during tension compared to compression, while only during compression a strong deformation-rate dependency was found. In fact, fetal lung tissue under compression showed clear viscoelastic features even for small strains. Thus, we propose that the fetal lung is much more vulnerable during inflation by mechanical ventilation compared to normal inspiration. Electrophysiological experiments with different hydrostatic pressure gradients acting on primary fetal distal lung epithelial cells revealed that the activity of the epithelial sodium channel (ENaC) and the sodium-potassium pump (Na,K-ATPase) dropped during pressures of 30 cmH2O. Thus, pressures used during mechanical ventilation might impair alveolar fluid clearance important for normal lung function.

Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway.

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na+ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na+ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na+ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na+ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na+ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na+ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na+ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway.

Epidermal growth factor strongly affects epithelial Na+ transport and barrier function in fetal alveolar cells, with minor sex-specific effects

Male sex remains an independent risk factor for respiratory distress syndrome (RDS) in preterm infants. Insufficient Na+ transport-mediated alveolar fluid clearance contributes to RDS development and we previously demonstrated sex-specific differences in Na+ transport. The epidermal growth factor (EGF) is important during fetal lung development with possible influence on Na+ transport. Sex-specific effects of EGF during surfactant synthesis were shown. We thus determined whether EGF exerts sex-specific effects on Na+ transport in fetal alveolar cells. We analyzed sex-specific fetal distal lung epithelial (FDLE) cells exposed to EGF and related ligands with Ussing chambers, RT-qPCR and Western blots. EGF strongly reduced the epithelial Na+ channel (ENaC) mRNA levels in both male and female FDLE cells. This was corroborated by a markedly reduced ENaC activity, while amiloride-insensitive pathways as well as barrier function were raised by EGF. In contrast to chronic effects, acute effects of EGF were sex-specific, because Na+ transport was reduced only in males. AKT phosphorylation was elevated only in female cells, while pERK1/2 was increased in both male and female cells. EGF showed certain sex- and time-dependent effects in FDLE cells. Nevertheless, the results suggest that EGF is an unlikely cause for the sex-specific differences in Na+ transport.

Paracrine stimulation of perinatal lung functional and structural maturation by mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) were shown to harbor therapeutic potential in models of respiratory diseases, such as bronchopulmonary dysplasia (BPD), the most common sequel of preterm birth. In these studies, cells or animals were challenged with hyperoxia or other injury-inducing agents. However, little is known about the effect of MSCs on immature fetal lungs and whether MSCs are able to improve lung maturity, which may alleviate lung developmental arrest in BPD.MethodsWe aimed to determine if the conditioned medium (CM) of MSCs stimulates functional and structural lung maturation. As a measure of functional maturation, Na+ transport in primary fetal distal lung epithelial cells (FDLE) was studied in Ussing chambers. Na+ transporter and surfactant protein mRNA expression was determined by qRT-PCR. Structural maturation was assessed by microscopy in fetal rat lung explants.ResultsMSC-CM strongly increased the activity of the epithelial Na+ channel (ENaC) and the Na,K-ATPase as well as their mRNA expression. Branching and growth of fetal lung explants and surfactant protein mRNA expression were enhanced by MSC-CM. Epithelial integrity and metabolic activity of FDLE cells were not influenced by MSC-CM. Since MSC’s actions are mainly attributed to paracrine signaling, prominent lung growth factors were blocked. None of the tested growth factors (VEGF, BMP, PDGF, EGF, TGF-β, FGF, HGF) contributed to the MSC-induced increase of Na+ transport. In contrast, inhibition of PI3-K/AKT and Rac1 signaling reduced MSC-CM efficacy, suggesting an involvement of these pathways in the MSC-CM-induced Na+ transport.ConclusionThe results demonstrate that MSC-CM strongly stimulated functional and structural maturation of the fetal lungs. These effects were at least partially mediated by the PI3-K/AKT and Rac1 signaling pathway. Thus, MSCs not only repair a deleterious tissue environment, but also target lung cellular immaturity itself.

Glucocorticoids Equally Stimulate Epithelial Na+ Transport in Male and Female Fetal Alveolar Cells.

Preterm infants frequently suffer from respiratory distress syndrome (RDS), possibly due to lower expression of epithelial Na+ channels (ENaC). RDS incidence is sex-specific, affecting males almost twice as often. Despite the use of antenatal glucocorticoids (GCs), the sex difference persists. It is still controversial whether both sexes benefit equally from GCs. We previously showed that Na+ transport is higher in female compared with male fetal distal lung epithelial (FDLE) cells. Since GCs increase Na+ transport, we hypothesized that their stimulating effect might be sex-specific. We analyzed FDLE cells with Ussing chambers and RT-qPCR in the presence or absence of fetal serum. In serum-free medium, GCs increased the ENaC activity and mRNA expression, independent of sex. In contrast, GCs did not increase the Na+ transport in serum-supplemented media and abolished the otherwise observed sex difference. Inhibition of the GC receptor in the presence of serum did not equalize Na+ transport between male and female cells. The GC-induced surfactant protein mRNA expression was concentration and sex-specific. In conclusion, female and male FDLE cells exhibit no sex difference in response to GCs with regard to Na+ transport, and GR activity does not contribute to the higher Na+ transport in females.

Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development.

Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear sub-population of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3′ sites. In FDLE cultured at the PO2 of the fetal lung, FGF-10 relieved the Spry2 interaction at the HRE region by promoting clearance of a 39 kDa form and this was accompanied by histone-3 S10K14 phosphoacetylation, promoter de-methylation, hypoxia inducible factor-1α activation and VEGF-A expression. This repressive characteristic of nuclear Spry2 was relieved in 16HBE14o- by shRNA knockdown, and stable expression of mutants (C218A; C221A) that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung.

Sex-specific effects of sex steroids on alveolar epithelial Na+ transport.

Alveolar fluid clearance mediates perinatal lung transition to air breathing in newborn infants, which is accomplished by epithelial Na+ channels (ENaC) and Na-K-ATPase. Male sex represents a major risk factor for developing respiratory distress, especially in preterm infants. We previously showed that male sex is associated with reduced epithelial Na+ transport, possibly contributing to the sexual dimorphism in newborn respiratory distress. This study aimed to determine sex-specific effects of sex steroids on epithelial Na+ transport. The effects of testosterone, 5α-dihydrotestosterone (DHT), estradiol, and progesterone on Na+ transport and Na+ channel expression were determined in fetal distal lung epithelial (FDLE) cells of male and female rat fetuses by Ussing chamber and mRNA expression analyses. DHT showed a minor effect only in male FDLE cells by decreasing epithelial Na+ transport. However, flutamide, an androgen receptor antagonist, did not abolish the gender imbalance, and testosterone lacked any effect on Na+ transport in male and female FDLE cells. In contrast, estradiol and progesterone increased Na+ transport and Na+ channel expression especially in females, and prevented the inhibiting effect of DHT in males. Estrogen receptor inhibition decreased Na+ channel expression and eliminated the sex differences. In conclusion, female sex steroids stimulate Na+ transport especially in females and prevent the inhibitory effect of DHT in males. The ineffectiveness of testosterone suggests that Na+ transport is largely unaffected by androgens. Thus, the higher responsiveness of female cells to female sex steroids explains the higher Na+ transport activity, possibly leading to a functional advantage in females.

Male Sex is Associated with a Reduced Alveolar Epithelial Sodium Transport.

Respiratory distress syndrome (RDS) is the most frequent pulmonary complication in preterm infants. RDS incidence differs between genders, which has been called the male disadvantage. Besides maturation of the surfactant system, Na+ transport driven alveolar fluid clearance is crucial for the prevention of RDS. Na+ transport is mediated by the epithelial Na+ channel (ENaC) and the Na,K-ATPase, therefore potential differences in their expression or activity possibly contribute to the gender imbalance observed in RDS. Fetal distal lung epithelial (FDLE) cells of rat fetuses were separated by sex and analyzed regarding expression and activity of the Na+ transporters. Ussing chamber experiments showed a higher baseline short-circuit current (ISC) and amiloride-sensitive ΔISC in FDLE cells of female origin. In addition, maximal amiloride-sensitive ΔISC and maximal ouabain-sensitive ΔISC of female cells were higher when measured in the presence of a permeabilized basolateral or apical membrane, respectively. The number of FDLE cells per fetus recoverable during cell isolation was also significantly higher in females. In addition, lung wet-to-dry weight ratio was lower in fetal and newborn female pups. Female derived FDLE cells had higher mRNA levels of the ENaC- and Na,K-ATPase subunits. Furthermore, estrogen (ER) and progesterone receptor (PR) mRNA levels were higher in female cells, which might render female cells more responsive, while concentrations of placenta-derived sex steroids do not differ between both genders during fetal life. Inhibition of ER-β abolished the sex differences in Na+ transport and female cells were more responsive to estradiol stimulation. In conclusion, a higher alveolar Na+ transport, possibly attributable to a higher expression of hormone receptors in female FDLE cells, provides an explanation for the well known sex-related difference in RDS occurrence and outcome.

Mechanotransduction via TRPV4 regulates inflammation and differentiation in fetal mouse distal lung epithelial cells.

BackgroundMechanical ventilation plays a central role in the injury of premature lungs. However, the mechanisms by which mechanical signals trigger an inflammatory cascade to promote lung injury are not well-characterized. Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable mechanoreceptor channel has been shown to be a major determinant of ventilator-induced acute lung injury in adult models. However, the role of these channels as modulators of inflammation in immature lungs is unknown. In this study, we tested the hypothesis that TRPV4 channels are important mechanotransducers in fetal lung injury.MethodsExpression of TRPV4 in the mouse fetal lung was investigated by immunohistochemistry, Western blot and qRT-PCR. Isolated fetal epithelial cells were exposed to mechanical stimulation using the Flexcell Strain Unit and inflammation and differentiation were analyzed by ELISA and SP-C mRNA, respectively.ResultsTRPV4 is developmentally regulated in the fetal mouse lung; it is expressed in the lung epithelium and increases with advanced gestation. In contrast, in isolated epithelial cells, TRPV4 expression is maximal at E17-E18 of gestation. Mechanical stretch increases TRPV4 in isolated fetal epithelial cells only during the canalicular stage of lung development. Using the TRPV4 agonist GSK1016790A, the antagonist HC-067047, and the cytokine IL-6 as a marker of inflammation, we observed that TRPV4 regulates release of IL-6 via p38 and ERK pathways. Interestingly, stretch-induced differentiation of fetal epithelial cells was also modulated by TRPV4.ConclusionThese studies demonstrate that TRPV4 may play an important role in the transduction of mechanical signals in the fetal lung epithelium by modulating not only inflammation but also the differentiation of fetal epithelial cells.

Role of GABA Receptors in Fetal Lung Development in Rats

Fluid accumulation is critical for lung distension and normal development. The multi-subunit γ-amino butyric acid type A receptors (GABAA) mainly act by mediating chloride ion (Cl−) fluxes. Since fetal lung actively secretes Cl−-rich fluid, we investigated the role of GABAA receptors in fetal lung development. The physiological ligand, GABA, and its synthesizing enzyme, glutamic acid decarboxylase, were predominantly localized to saccular epithelium. To examine the effect of activating GABAA receptors in fetal lung development in vivo, timed-pregnant rats of day 18 gestation underwent an in utero surgery for the administration of GABAA receptor modulators into the fetuses. The fetal lungs were isolated on day 21 of gestation and analyzed for changes in fetal lung development. Fetuses injected with GABA had a significantly higher body weight and lung weight when compared to phosphate-buffered saline (control)-injected fetuses. GABA-injected fetal lungs had a higher number of saccules than the control. GABA increased the number of alveolar epithelial type II cells as indicated by surfactant protein C-positive cells. However, GABA decreased the number of α-smooth muscle actin-positive myofibroblasts, but did not affect the number of Clara cells or alveolar type I cells. GABA-mediated effects were blocked by the GABAA receptor antagonist, bicuculline. GABA also increased cell proliferation and Cl− efflux in fetal distal lung epithelial cells. In conclusion, our results indicate that GABAA receptors accelerate fetal lung development, likely through an enhanced cell proliferation and/or fluid secretion.

Control of HIF-1α and vascular signaling in fetal lung involves cross talk between mTORC1 and the FGF-10/FGFR2b/Spry2 airway branching periodicity clock

Lung development requires coordinated signaling between airway and vascular growth, but the link between these processes remains unclear. Mammalian target of rapamycin complex-1 (mTORC1) can amplify hypoxia-inducible factor-1α (HIF-1α) vasculogenic activity through an NH2-terminal mTOR binding (TOS) motif. We hypothesized that this mechanism coordinates vasculogenesis with the fibroblast growth factor (FGF)-10/FGF-receptor2b/Spry2 regulator of airway branching. First, we tested if the HIF-1α TOS motif participated in epithelial-mesenchymal vascular signaling. mTORC1 activation by insulin significantly amplified HIF-1α activity at fetal Po2 (23 mmHg) in human bronchial epithelium (16HBE14o-) and induced vascular traits (Flk1, sprouting) in cocultured human embryonic lung mesenchyme (HEL-12469). This enhanced activation of HIF-1α by mTORC1 was abolished on expression of a HIF-1α (F99A) TOS-mutant and also suppressed vascular differentiation of HEL-12469 cocultures. Next, we determined if vasculogenesis in fetal lung involved regulation of mTORC1 by the FGF-10/FGFR2b/Spry2 pathway. Fetal airway epithelium displayed distinct mTORC1 activity in situ, and its hyperactivation by TSC1−/− knockout induced widespread VEGF expression and disaggregation of Tie2-positive vascular bundles. FGF-10-coated beads grafted into fetal lung explants from Tie2-LacZ transgenic mice induced localized vascular differentiation in the peripheral mesenchyme. In rat fetal distal lung epithelial (FDLE) cells cultured at fetal Po2, FGF-10 induced mTORC1 and amplified HIF-1α activity and VEGF secretion without induction of ERK1/2. This was accompanied by the formation of a complex between Spry2, the cCBL ubiquitin ligase, and the mTOR repressor, TSC2, which abolished GTPase activity directed against Rheb, the G protein inducer of mTORC1. Thus, mTORC1 links HIF-1α-driven vasculogenesis with the FGF-10/FGFR2b/Spry2 airway branching periodicity regulator.

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

Variations in ENaC subunit composition may determine amiloride sensitivity and β-adrenergic stimulation of lung fluid absorption

reabsorption of fetal lung fluid at birth and resolution of alveolar edema in the adult lung are driven by active sodium and chloride transport across the alveolar epithelial barrier (for review see Ref. [8][1]). The rate of fetal lung fluid absorption and alveolar edema removal can be increased by

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

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.

GABAA receptors play a role in fluid secretion in rat fetal lung

Fluid secretion in the fetal lung is driven by Cl−secretion and is essential for lung development. However, the mechanism of Cl− and fluid secretion in the fetal lung is not completely clear. GABAA receptors are ligand-gated Cl−channels. We show that native GABAA receptors in fetal distal lung epithelial cells are composed of α3-, α4-, β1-, β2-, and π-subunits by co-immunoprecipitation. GABA increased lumen size and Cl− concentration on fetal lung organ culture while GABA receptor antagonists, picrotoxin and biccuculine had an opposite effect. The knock-down of GABAA receptor π subunit by RNA interference also inhibited the fluid secretion. In vitro phosphorylation studies denoted that only β1-subunit of GABAA receptors was phosphorylated on serine residue and this phosphorylation was enhanced in the fetal distal lung epithelial cells stimulated with PMA. Inhibitors of protein kinase C, phospholipase C and P2Y receptor all reduced lumen size in the presence of GABA and inhibited the phosphorylation of the β1-subunit on fetal lung organ culture. Taken together, our results suggest that fetal distal lung epithelial cells contain functional ionotropic GABAA receptors. These receptors could participate in fluid secretion in the fetal lung and this process is regulated by protein phosphorylation (supported by NIH HL-052146, HL-071628 and March of Dimes to LL).

Oxygen and Glucocorticoids Modulate αENaC mRNA Translation in Fetal Distal Lung Epithelium

Glucocorticoid hormones play an important role in fetal lung maturation. It is unknown how they interact with changes in O2 tension, which play an important role in converting the lung from a fluid-secreting to a fluid-absorbing organ at birth. Airspace fluid absorption arises from active transepithelial Na+ transport with the amiloride-sensitive epithelial Na channel (ENaC), consisting of alpha, beta, and gamma subunits, representing the rate-limiting step under nonpathologic conditions. We investigated the individual and combined effects of dexamethasone (DEX) and PO2 on alphaENaC mRNA levels, rate of alphaENaC protein synthesis, and amiloride-sensitive short-circuit current in primary cultures of rat fetal distal lung epithelial cells. DEX significantly induced alphaENaC mRNA in fetal (3%) and postnatal (21%) O2, but increases in alphaENaC protein synthesis and function occurred only when epithelia were grown under a postnatal PO2. Sucrose density gradient analyses showed that DEX treatment of cells cultured at 3% O2 decreased the association of alphaENaC mRNA with large polysomes and enhanced the association with small polysomes. Conversely, incubation of DEX-treated cells in 21% O2 restored alphaENaC mRNA association with large polysomes. No significant changes were seen in the overall polyribosome profiles or in the distribution of mRNAs encoding beta and gamma subunits of ENaC or cytokeratin 18, indicating specific modulation of alphaENaC mRNA translation. These data suggest that postnatal O2 exposure may be important for efficient translation of the alphaENaC mRNA.

Postnatal glucocorticoids induce alpha-ENaC formation and regulate glucocorticoid receptors in the preterm rabbit lung.

At birth, lung fluid clearance is coupled to Na+ transport through epithelial Na+ channels (ENaC) in the distal lung epithelium. We evaluated the effect of postnatal glucocorticoids (GC) on lung alpha-ENaC expression in preterm 29-day gestational age (GA) fetal rabbits. Postnatal treatment of 29-day GA fetuses with 0.5 mg/kg of dexamethasone (Dex) iv resulted in a 2- and 22-fold increase in lung alpha-ENaC mRNA expression compared with saline-treated fetuses after 8 and 16 h, respectively. Lung alpha-ENaC protein levels in Dex-treated fetuses were also elevated compared with saline-treated counterparts. The extravascular lung water (EVLW)/dry lung tissue weight ratios of 29-day GA fetuses treated with either saline or Dex decreased over 24 h compared with that observed at birth; however, at 24 h, the EVLW/dry lung tissue weight ratios of saline- and Dex-treated fetuses were similar. Dex-induced alpha-ENaC mRNA and protein levels were attenuated by glucocorticoid receptor (GCR) antagonist RU-486 in fetal distal lung epithelial cells isolated from 29-day GA fetuses, indicating that GC-dependent augmentation of lung alpha-ENaC requires the presence of functional GCR. Lung GCR mRNA expression and protein levels were elevated in 29-day GA fetuses compared with fetuses at earlier GA. Exposure of 29-day GA fetuses to Dex for 16 h caused a 2.1-fold increase in lung GCR mRNA expression, but GCR protein levels were decreased in Dex-treated fetuses after 24 h. We conclude that postnatal treatment of preterm 29-day GA fetal rabbits with GC results in an elevation of lung alpha-ENaC accompanied by an autoregulation of pulmonary GCR.

Hormonal modulation of Na(+) transport in rat fetal distal lung epithelial cells.

Isolated rat fetal distal lung epithelial (FDLE) cells were cultured (approximately 48 h) on permeable supports in medium devoid of hormones and growth factors whilst P(O2) was maintained at the level found in either the fetal (23 mmHg) or the postnatal (100 mmHg) alveolar regions. The cells became incorporated into epithelial layers that generated a basal short-circuit current (I(SC)) attributable to spontaneous Na(+) absorption. Cells at neonatal P(O2) generated larger currents than did cells at fetal P(O2), indicating that this Na(+) transport process is oxygen sensitive. Irrespective of P(O2), isoprenaline failed to elicit a discernible change in I(SC), demonstrating that beta-adrenoceptor agonists do not stimulate Na(+) transport under these conditions. However, isoprenaline did elicit cAMP accumulation in these cells, indicating that functionally coupled beta-adrenoceptors are present. Further experiments showed that isoprenaline did increase I(SC) in cells treated (24 h) with a combination of tri-iodothyronine (T(3), 10 nM) and dexamethasone (200 nM). Studies of basolaterally permeabilised cells showed that these hormones are essential for the isoprenaline-evoked increase in the apical membrane's Na(+) conductance (G(Na)), whereas isoprenaline-evoked changes in apical Cl(-) conductance (G(Cl)) can occur in both control and hormone-treated cells. Irrespective of their hormonal status, FDLE cells thus express beta-adrenoceptors that are functionally coupled to adenylate cyclase, and allow beta-adrenoceptor agonists to modulate the apical membrane's anion conductance. However, T(3) and dexamethasone are needed if these receptors are to exert control over G(Na). These hormones may thus play an important role in the functional maturation of the lung by allowing beta-adrenoceptor-mediated control over epithelial Na(+) channels in the apical plasma membrane.

CAMP regulation of Cl(-) and HCO(-)(3) secretion across rat fetal distal lung epithelial cells.

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (I(sc)) and transepithelial resistances were 7.9 +/- 0.5 microA/cm(2) and 1,018 +/- 73 Omega.cm(2), respectively (means +/- SE; n = 12). Apical amiloride (10 microM) inhibited basal I(sc) by approximately 50%. Subsequent addition of forskolin (10 microM) increased I(sc) from 3.9 +/- 0.63 microA/cm(2) to 7.51 +/- 0.2 microA/cm(2) (n = 12). Basolateral bumetanide (100 microM) decreased forskolin-stimulated I(sc) from 7.51 +/- 0.2 microA/cm(2) to 5.62 +/- 0.53, whereas basolateral 4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of HCO secretion, blocked the remaining I(sc). Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl(-)- or HCO(-)(3)-free solutions; however, no response was seen using HCO(-)(3)- and Cl(-)-free solutions. The forskolin-stimulated I(sc) was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I(sc) across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl(-) and HCO(-)(3) secretion across rat FDLE cells mediated via CFTR.