Fetal Distal Lung Epithelium Research Articles

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.

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.

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.

The effect of hyperoxia on fluid transport by fetal distal lung epithelia

Objective To investigate the effect of hyperoxia on fluid transport by fetal distal lung epithelia(FDLE) and the expression of epithelial sodium channel(ENaC) in these cells.Methods FDLE were isolated and randomized into hyperoxia group and normoxia group,which were primarily cultured under hyperoxic or normoxic conditions,respectively.Fluid transport was measured using monolayers of FDLE cultured on Transwell permeable inserts.Western blot was applied to examine the α-ENaC expression.Results Fluid transport across monolayer of FDLE was increased in cells exposed to hyperoxia compaired with cells cultured in normoxic conditions (1.78 ± 0.19 vs 1.06 ± 0.11,P ＜ 0.001).Amiloride significantly decreased the fluid transport in both of the hyperoxia and normoxia groups,but in the presence of amiloride there were no difference between the two groups.The expression of α-ENaC was inhibited by hyperoxia to some extent(24h:0.44 ±0.04 vs 0.40 ±0.04,P=0.22; 48h:0.35 ±0.03 vs 0.47 ±0.06,P =0.03).Conclusion Hyperoxia enhanced total and amiloride-sensitive fluid transport by FDLE.However,the expression of α-ENaC decreased in these cells. Key words: Hyperoxia; Fetal distal lung epithelia; Fluid transport; Epithelial sodium channel

Benzimidazolones enhance the function of epithelial Na+ transport

Pharmacological enhancement of vectorial Na⁺ transport may be useful to increase alveolar fluid clearance. Herein, we investigated the influence of the benzimidazolones 1-ethyl-1,3-dihydro-2-benzimidazolone (1-EBIO), 5,6-dichloro-1-EBIO (DC-EBIO) and chlorzoxazone on vectorial epithelial Na⁺ transport. Effects on vectorial Na⁺ transport and amiloride-sensitive apical membrane Na⁺ permeability were determined by measuring short-circuit currents (I(SC)) in rat fetal distal lung epithelial (FDLE) monolayers. Furthermore, amiloride-sensitive membrane conductance and the open probability of epithelial Na⁺ channels (ENaC) were determined by patch clamp experiments using A549 cells. I(SC) was increased by approximately 50% after addition of 1-EBIO, DC-EBIO and chlorzoxazone. With permeabilized basolateral membranes in the presence of a 145:5 apical to basolateral Na⁺ gradient, the benzimidazolones markedly increased amiloride-sensitive I(SC). 5-(N-Ethyl-N-isopropyl)amiloride-induced inhibition of I(SC) was not affected. The benzamil-sensitive I(SC) was increased in benzimidazolone-stimulated monolayers. Pretreating the apical membrane with amiloride, which inhibits ENaC, completely prevented the stimulating effects of benzimidazolones on I(SC). Furthermore, 1-EBIO (1 mM) and DC-EBIO (0.1 mM) significantly increased (threefold) the open probability of ENaC without influencing current amplitude. Whole cell measurements showed that DC-EBIO (0.1 mM) induced an amiloride-sensitive increase in membrane conductance. Benzimidazolones have a stimulating effect on vectorial Na⁺ transport. The antagonist sensitivity of this effect suggests the benzimidazolones elicit this action by activating the highly selective ENaC currents. Thus, the results demonstrate a possible new strategy for directly enhancing epithelial Na⁺ transport.

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.

Long-term terbutaline exposure stimulates α1-Na+-K+-ATPase expression at posttranscriptional level in rat fetal distal lung epithelial cells

Transepithelial Na(+) transport through epithelial Na(+) channels (ENaC) on the apical membrane and Na(+)-K(+)-ATPase activity on the basolateral membrane of distal lung epithelial cells are critical for alveolar fluid clearance. Acute exposure to beta-adrenergic agonists stimulates lung fluid clearance by increasing Na(+) transport. We investigated the effects of chronic exposure to the beta(2)-adrenergic agonist terbutaline on the transepithelial Na(+) transport in rat fetal distal lung epithelia (FDLE). FDLE monolayers exposed to 10(-4) M terbutaline for 48 h had significantly increased propanolol-blockable transepithelial total and amiloride-sensitive short-circuit current (I(sc)); however, when these chronically exposed monolayers were acutely exposed to additional beta-agonists and intracellular cAMP upregulators, there was no further increase in I(sc). Monolayers exposed to terbutaline for >48 h had I(sc) similar to control cells. Ouabain-sensitive Na(+)-K(+)-ATPase activity was increased in 48-h terbutaline-exposed FDLE whose apical membranes were permeabilized with nystatin. In contrast, terbutaline did not increase amiloride-sensitive apical membrane I(sc) in FDLE whose basolateral membranes were permeabilized with nystatin. Terbutaline treatment did not affect alpha-, beta-, or gamma-ENaC mRNA or alpha-ENaC protein steady-state levels, but increased total cellular levels and rate of synthesis of alpha(1)-Na(+)-K(+)-ATPase protein in FDLE in the absence of any change in alpha(1)-Na(+)-K(+)-ATPase mRNA. Total cellular beta(1)-Na(+)-K(+)-ATPase mRNA and protein levels were not affected by terbutaline. These data suggest that FDLE have different responses from adult type II epithelial cells when chronically exposed to terbutaline, and their increased transepithelial Na(+) transport occurs via a posttranscriptional increase in alpha(1)-Na(+)-K(+)-ATPase expression.

Glucocorticoid-Mediated Repression of REDD1 mRNA Expression in Rat Fetal Distal Lung Epithelial Cells

REDD1 (Regulated in Development and DNA Damage-1) is a stress-response gene that represses mammalian target of rapamycin (mTOR) thus decreasing protein synthesis. In contrast to studies using cell lines and adult alveolar type II (ATII) cells, we find that REDD1 mRNA levels did not increase in rat fetal distal lung epithelia (FDLE) or fetal lung fibroblasts grown in primary cultures and then exposed to 3% O2. REDD1 mRNA expression was repressed by dexamethasone (DEX) in FDLE and ATII, but induced by DEX in fibroblasts. Lung epithelial cell lines, A549 and MLE-15, showed increases in REDD1 mRNA in response to hypoxia and DEX. The effect of DEX on REDD1 mRNA and protein in FDLE and fibroblasts was dose- and time-dependent. Inhibitor studies support repression of REDD1 mRNA by DEX in FDLE was mediated via glucocorticoid receptor and not by nongenomic effects of glucocorticoids via MAPK pathways. The half-life of REDD1 mRNA was shorter in DEX-exposed FDLE compared with hormone-free media suggesting that DEX reduced REDD1 mRNA stability in FDLE. These studies indicate that REDD1 expression in response to hypoxia and DEX is cell-type specific and that physiologically appropriate levels of PO2 should be used when investigating fetal lung development.

Pulmonary Neuroendocrine Cell-Secreted Factors May Alter Fetal Lung Liquid Clearance

The neuroendocrine system is most active at birth and may play a role in the transition from fetal to postnatal life, in particular in the lungs' transition from fluid secretion to fluid absorption. Pulmonary neuroendocrine cells do release dopamine (DA), serotonin, and gastrin-releasing peptide but their effects on lung ion and fluid transport are poorly understood. Therefore, we studied their effects on fetal distal lung explants and primary cultures of fetal distal lung epithelium (FDLE). We show that DA, but neither serotonin nor gastrin-releasing peptide, alters ion and fluid transport, in a dose-dependent manner. DAs effects were abrogated by D1/D2 receptor blockers in FDLE but not in explants. Propranolol abrogated DAs effects in both models. DA increased intracellular cAMP levels in FDLE. Terbutaline, forskolin, and isobutylmethylxanthine did not increase short circuit current (Isc) in DA-treated cells, despite a further increase in cAMP. We conclude that at least one, but not all mediators released by pulmonary neuroendocrine cells alter distal lung epithelial ion transport.

Amiloride-insensitive Na+and fluid absorption in the mammalian distal lung

The ability of the distal lung epithelia to actively transport Na+, with Cl- and water following, from the alveolar spaces inversely correlates with morbidity and mortality of infants, children, and adults with alveolar pulmonary edema. It is now recognized, in contrast to many other Na+ transporting epithelia, that at least half of this active transport is not sensitive to amiloride, which inhibits the epithelial Na+ channel. This paper reviews amiloride-insensitive Na+ and fluid transport in the mammalian distal lung unit under basal conditions and speculates on potential explanations for this amiloride-insensitive transport. It also provides new information, using primary cultures of rat fetal distal lung epithelia and alveolar type II cells grown under submersion and air-liquid interface culture conditions, regarding putative blockers of this transport.

Effects of cardiogenic edema fluid on ion and fluid transport in the adult lung

We have previously shown that cardiogenic pulmonary edema fluid (EF) increases Na(+) and fluid transport by fetal distal lung epithelia (FDLE) (Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM and O'Brodovich H. J Physiol 544: 537-548, 2002). We now report the effect of EF on Na(+) and fluid transport by the adult lung. We first studied primary cultures of adult type II (ATII) epithelium and found that overnight exposure to EF increased Na(+) transport, and this effect was mainly due to factors other than catecholamines. Plasma did not stimulate Na(+) transport in ATII. Purification of EF demonstrated that at least some agent(s) responsible for the amiloride-insensitive component resided within the globulin fraction. ATII exposed to globulins demonstrated a conversion of amiloride-sensitive short-circuit current (I(sc)) to amiloride-insensitive I(sc) with no increase in total I(sc). Patch-clamp studies showed that ATII exposed to EF for 18 h had increased the number of highly selective Na(+) channels in their apical membrane. In situ acute exposure to EF increased the open probability of Na(+)-permeant ion channels in ATII within rat lung slices. EF did increase, by amiloride-sensitive pathways, the alveolar fluid clearance from the lungs of adult rats. We conclude that cardiogenic EF increases Na(+) transport by adult lung epithelia in primary cell culture, in situ and in vivo.

Steroid and Oxygen Effects on eIF4F Complex, mTOR, and ENaC Translation in Fetal Lung Epithelia

Fetal distal lung epithelium (FDLE) must increase amiloride-sensitive epithelial Na(+) channel (ENaC) activity during the perinatal period to increase Na(+) transport and fluid clearance. Glucocorticosteroid (GC) levels increase, there is a 7-fold increase in Po(2) at birth, and we have previously shown that dexamethasone (DEX)-induced alpha-ENaC mRNA is efficiently translated only under postnatal (21%) O(2) (Otulakowski et al., AJRCMB 2006;34:204-212). Translation of mRNAs with long GC-rich 5'UTRs, such as alpha-ENaC mRNA, are sensitive to the amount of eIF4F, the mRNA 5'-cap binding complex composed of eIF4E and eIF4G. We now show, by Western blotting and m(7)GTP-Sepharose pull-down experiments, that in FDLE cultured under 3% O(2), DEX decreases formation of eIF4F and increases association of eIF4E with its inhibitor 4E-BP by changing 4E-BP phosphorylation. Conversely, FDLE cultured at 21% O(2) expressed lower levels of 4E-BP and maintained eIF4E-eIF4G association independent of DEX. Phosphorylation of 4E-BP is regulated by the kinase mTOR. Under 3% O(2), DEX decreased abundance of phosphorylated forms of the mTOR effectors, S6 kinase and ribosomal protein S6. Neither effect was associated with changes in REDD1, an upstream regulator of mTOR. When mTOR was inhibited (3 nM rapamycin) there was reduced 4E-BP phosphorylation, fewer ribosomes on alpha-ENaC mRNA, and decreased amiloride-sensitive short-circuit current, but no change in ribosomal loading onto any of beta- or gamma-ENaC or cytokeratin 18 mRNAs. We speculate that at birth increased Po(2) acts with GC through an mTOR-related pathway to increase alpha-ENaC protein synthesis, thereby promoting lung fluid absorption.

The role of α-, β-, and γ-ENaC subunits in distal lung epithelial fluid absorption induced by pulmonary edema fluid

Edema fluid (EF) increases epithelial Na(+) transport by rat fetal distal lung epithelia (FDLE) and induces net lung fluid absorption in fetal mouse lung explants [Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. J Physiol (Lond) 544: 537-548, 2002]. We now show that EF increases fluid absorption across monolayers of rat FDLE in a dose-dependent manner. To study the role of subunits of the epithelial Na(+) channel (ENaC) in the phenomena, we cultured explants from the distal lungs of 16-day gestational age wild-type (WT) or alpha-, beta-, or gamma-ENaC knockout or heterozygote (HT) mice. WT explants cultured in media continuously expanded over time as a result of net fluid secretion. In contrast, when explants were exposed to EF for 24 h, net fluid absorption occurred. EF-exposed explants had normal histology, but marked changes were seen after Triton X-100 or staurosporine exposure. Transmission electron microscopy showed EF promoted lamellar body formation and abundant surfactant in the explants' lumens. EF-induced changes in explant size were similar in alpha-ENaC knockout, WT, and HT littermate fetal lung explants (P > 0.05). In contrast, EF's effect was attenuated in beta- and gamma-ENaC knockouts (P < 0.05) vs. WT and HT littermate fetal lung explants. EF exposure slightly decreased or had no effect on mRNA levels for alpha-ENaC in various mouse genotypes but decreased expression of beta- and gamma-ENaC subunit mRNAs (P < 0.01) across all genotype groups. We conclude that beta- and gamma-, but not alpha-, ENaC subunits are essential for EF to exert its maximal effect on net fluid absorption by distal lung epithelia.

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

Oxygen and dexamethasone modulate αENaC translation in fetal lung epithelia via availability of eIF4F

At birth, fetal distal lung epithelia (FDLE) must rapidly upregulate Na+ transport, the rate limiting step of which is the amiloride-sensitive epithelial Na+ channel (ENaC). Although glucocorticoids (GC) mature the fetal lung it is unknown how GC interact with pO2 at birth to induce lung fluid absorption. We found that dexamethasone (DEX)-induced αENaC mRNA was efficiently translated into protein in primary cultures of FDLE only under postnatal (21%) O2 (Otulakowski et al., AJRCMB;in press). Sucrose density gradient analyses of polysomes showed changes in αENaC mRNA distribution consistent with specific modulation of αENaC translation initiation; ie. DEX treatment of cells under fetal (3%) O2 decreased association of αENaC mRNA with large polysomes, while shifting DEX-treated cells to 21% O2 restored association with large polysomes. αENaC mRNA possesses a long 5′UTR with a high GC content near the 5′-cap, typical of mRNAs sensitive to limiting availability of eIF4F, the mRNA 5′-cap binding complex composed of eIF4E and eIF4G. Western blots and m7GTP-Sepharose pull-down experiments showed DEX decreased formation of eIF4F and increased association of eIF4E with its inhibitor 4E-BP in FDLE cultured at 3% O2, mediated by changes in 4E-BP phosphorylation. Conversely, FDLE cultured at 21% O2 expressed lower levels of 4E-BP and maintained eIF4E-eIF4G association in the presence of DEX. This suggests that the 7-fold increase in pO2 acts synergistically with GC to prepare the newborn lung to actively absorb fetal lung liquid. Supported by CIHR.

Clearance of Fluid From Airspaces of Newborns and Infants

After completing this article, readers should be able to: 1. Describe fetal lung fluid secretion, its application on lung development, and its reabsorption immediately after birth. 2. List the primary cellular mechanism responsible for lung fluid absorption. 3. Describe clinical conditions in newborns and infants associated with impaired sodium transport. 4. Describe how sodium transport can be influenced by fetal lung maturity. When the airspaces of the lungs are filled with fluid, normal gas exchange cannot take place, and there is marked respiratory distress and the need for intensive care. This occurs in only two situations: failure to clear fetal lung liquid at birth and leakage of fluid from the vasculature filling the alveoli (alveolar pulmonary edema). Fluid that is secreted into the fetal airspaces to enable normal lung development must be absorbed at the time of birth for a normal transition from fetal to postnatal life. Failure to do so can lead to two well-known clinical syndromes: transient tachypnea of the newborn (TTN) and when there is coexistent relative surfactant deficiency, neonatal respiratory distress syndrome (nRDS). Pulmonary edema can be caused postnatally by many disorders; those that occur frequently during the neonatal period are patent ductus arteriosus or structural congenital heart disease with marked left-to-right shunting. It is not only biologically reasonable to assume that clearance of such airspace fluid would be beneficial to the patient, but studies in adults suffering from pulmonary edema have shown that the lung’s ability to clear such fluid correlates with survival. In this article, we review how the epithelium of the lung uses the active transport of sodium (Na+), followed by chloride (Cl−) and water, from the apical to basolateral side of the alveolar lining cells to clear fluid and how the underlying mechanisms for active Na+ transport are influenced by the degree of …

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.

Beta-adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia.

Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current (I(sc)) that was inhibited (~80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na(+) from the apical solution. Isoprenaline increased the amiloride-sensitive I(sc) about twofold. Experiments in which apical membrane Na(+) currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na(+) conductance (G(Na(+))). Isoprenaline also increased apical Cl- conductance (G(Cl-)) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The isoprenaline-evoked changes in G(Na(+)) and G(Cl(minus sign)) could account for the changes in I(sc) observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of I(sc) or G(Na(+)) demonstrating that the rise in G(Cl-) is not essential to the stimulation of Na(+) transport.