The adult mature fetal, but not immature fetal, lung is capable of actively transporting Na+ from the alveolar space. The reason for the impaired Na+ transport in the immature lung is not known; however, the apical membrane Na+ channel is the rate-limiting step for epithelial Na+ transport. This study determined whether transcripts coding for the adult rat colonic epithelial Na+ channel (alpha rENaC) were present in the fetal and adult lung and whether they were developmentally regulated. Similarly sized alpha rENaC transcripts were identified in RNA isolated from fetal and adult whole rat lung, primary cultures of fetal and adult alveolar epithelium, and adult rat whole kidneys, suggesting that the lung alpha rENaC is a similar transcript to that found in the salt-deprived rat colonic epithelium. There were low mRNA levels in 17- to 18-day gestational age (GA) fetal lungs and epithelium (term GA = 22 days), but these levels increased markedly during the saccular stage of lung development (20 days GA) and remained high in adult lungs. The combined administration of thyroid-releasing hormone and dexamethasone to pregnant rats between 16 and 18 days GA induced the expression of lung alpha rENaC in their fetuses. We conclude that alpha rENaC is expressed in mature fetal and adult alveolar epithelium and that it is influenced by hormones known to alter maturation of the fetal lung.

Cultured rat fetal distal lung epithelial cells (FDLEs), when switched from fetal (3%) to postnatal (21%) O2 concentrations, have increased epithelial Na+ channel (ENaC) mRNA levels and amiloride-sensitive Na+transport [O. Pitkänen, A. K. Tanswell, G. Downey, and H. O’Brodovich. Am. J. Physiol. 270 ( Lung Cell. Mol. Physiol. 14): L1060–L1066, 1996]. The mechanisms by which O2 mediates these effects are unknown. After isolation, FDLEs were kept at 3% O2 overnight, then switched to 21% O2 (3–21% O2 group) or maintained at 3% O2 (3–3% O2 group) for 48 h. The amiloride-sensitive short-circuit current ( I sc) in the 3–21% O2 group was double that in the 3–3% O2 group. Amiloride-sensitive I sc could not be induced by medium conditioned by 21% O2-exposed FDLEs but was reversed by returning the cells to 3% O2. Neither the cyclooxygenase inhibitor ibuprofen, liposome-encapsulated catalase, nor hydroperoxide scavengers (U-74389G or Trolox) blocked the O2-induced amiloride-sensitive I sc. In contrast, the cell-permeable superoxide scavenger tetramethylpiperidine- N-oxyl (TEMPO) eliminated the O2-induced increases in amiloride-sensitive I sc and ENaC mRNA levels. The switch from 3 to 21% O2 induced the transcription factor nuclear factor-κB, which could also be blocked by TEMPO. We conclude that 1) the O2-induced increase in amiloride-sensitive I sc is reversible and 2) the O2-induced increase in amiloride-sensitive I sc and ENaC mRNA levels is associated with activation of nuclear factor-κB and may be mediated, at least in part, by superoxide.

The rat amiloride-sensitive epithelial sodium channel (rENaC) is the rate-limiting step for vectorial transport of Na+ across tight epithelia. The complex is composed of three subunits, alpha, beta, and gamma. Expression of the subunits has been shown to be tissue-specific and developmentally and hormonally regulated. To study mechanisms involved in transcriptional regulation of alpharENaC, we determined the genomic organization of the alpharENaC gene. By 5' rapid amplification of cDNA ends and primer extension, two transcriptional start sites were detected 453 base pairs (bp) apart, resulting in alternative 5' untranslated region (UTR) lengths of 515 or 62 bp. The longer 5' UTR is more prevalent in fetal lung than in adult lung or kidney. The 5' untranslated and coding regions are contained within 12 exons, with the translation start site located within the first exon. Sequence analysis of approximately 1,500 bp of 5' flanking DNA identified putative binding sites for transcription factors PEA3, SP1, AP-1, nuclear factor-kappaB, and thyroid and glucocorticoid receptors. alpharENaC promoter-reporter gene constructs produced low levels of reporter gene activity in transiently transfected cells, which could be increased by dexamethasone (DEX) treatment. Tri-iodothyronine treatment alone had no effect but potentiated stimulation by DEX.

To investigate the bioelectric properties on one of the cell types that line the distal lung unit, we isolated type II alveolar epithelium from 18- to 20-day gestation fetal rats (term = 22 days) and grew them on collagen-coated nitrocellulose filters. Amiloride impaired ion transport in a dose-dependent fashion (10(-4) to 10(-6) M) with 10(-4) M decreasing potential difference (PD) (mean +/- SE, 2.0 +/- 0.49 to 0.9 +/- 0.26 mV, P less than 0.01, lumen negative) and short-circuit current (Isc) (7.0 +/- 1.0 to 2.4 +/- 0.64 uA/cm2, P less than 0.01) without affecting resistance (R) (241 +/- 33 to 216 +/- 41 omega. cm2). Benzamil (10(-5) M) but not dimethylamiloride (10(-5) M) decreased Isc. Terbutaline (10(-3) M) increased PD from 1.2 +/- 0.13 to 3.3 +/- 0.40 mV (P less than 0.01), and application of amiloride (10(-4) M) after terbutaline reduced PD and Isc to less than initial base-line values. The Na(+)-K(+)-2Cl- cotransport inhibitors bumetanide (10(-4) M) and furosemide (10(-3) M) had no effect on PD and Isc either before or after terbutaline. Neither the Cl- channel blocker diphenylamine-2-carboxylate (10(-3) M) nor the Na(+)-glucose cotransport inhibitor phloridzin (10(-3) M) affected the bioelectric properties. Fetal type II alveolar epithelium in primary culture actively transport ions and, on the basis of inhibitor-agonist experiments, probably do not secrete Cl- but absorb Na+ through Na+ channels.

[537][538][539][540][541][542][543][544][545][546][547][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 ϩ channel (ENaC) in the phenomena, we cultured explants from the distal lungs of 16-day gestational age wild-type (WT) or ␣-, ␤-, or ␥-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 ␣-ENaC knockout, WT, and HT littermate fetal lung explants (P Ͼ 0.05). In contrast, EF's effect was attenuated in ␤-and ␥-ENaC knockouts (P Ͻ 0.05) vs. WT and HT littermate fetal lung explants. EF exposure slightly decreased or had no effect on mRNA levels for ␣-ENaC in various mouse genotypes but decreased expression of ␤-and ␥-ENaC subunit mRNAs (P Ͻ 0.01) across all genotype groups. We conclude that ␤-and ␥-, but not ␣-, ENaC subunits are essential for EF to exert its maximal effect on net fluid absorption by distal lung epithelia. epithelium; Na ϩ transport; lung development; amiloride-sensitive epithelial Na ϩ channel PULMONARY EDEMA can mainly occur as a result of one or both of the following mechanisms: increased transvascular pressure in the pulmonary microvasculature, as occurs in congestive heart failure, or increased permeability of the alveolar-capillary membrane to solutes, as occurs in adult respiratory distress syndrome. The ability of the lungs to clear this air space fluid has important clinical implications. For example, others have demonstrated that the active absorption of air space fluid in adults with pulmonary edema correlates with improved survival (20). Air space fluid clearance is actively driven by epithelial Na ϩ transport, which crosses the apical membrane and is then extruded across the basolateral membrane by Na ATPase (18, 19). The rate-limiting step in active Na ϩ transport by epithelia is the number and activity of the apical membrane Na ϩ channels. One of these Na ϩ channels is the amiloridesensitive epithelial Na ϩ channel (ENaC) that is composed of ␣-, ␤-, and ␥-subunits (6). However, it has been shown that normal mammalian lung epithelia have both an amiloridesensitive and an amiloride-insensitive component to their active Na ϩ transport both in vivo (24) and in vitro (8, 25). Presently, the molecular basis of this amiloride-insensitive Na ϩ transport is poorly understood. Regardless, if one could determine mechanisms underlying the regulation of this amiloride-sensitive and -insensitive Na ϩ transport, one could in the future identify novel approaches for the therapy of patients with pulmonary edema.It has been...

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current ( I sc) without affecting total I sc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the I sc. The effect was reversed by the addition of N-monomethyl-l-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

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.

The Na+ transport function of alveolar epithelium represents an important mechanism for air space fluid clearance after acute lung injury. We studied the effect of endotoxin-stimulated rat alveolar macrophages on lung epithelial ion transport and permeability in vitro. Cultured rat distal lung (alveolar) epithelial monolayers incubated with both endotoxin and macrophages demonstrated a 75% decline in transepithelial resistance and a selective 60% reduction in amiloride-sensitive short-circuit current (Isc). Single-channel patch-clamp analysis demonstrated a 60% decrease in the density of 25-pS nonselective cation (NSC) channels on the apical membrane of epithelium exposed to both endotoxin and macrophages. A concurrent reduction in epithelial F-actin content suggested a role for actin depolymerization in mediating this effect. Incubation of cocultures with the methylated L-arginine (Arg) derivative NG-monomethyl-L-arginine prevented the reduction in epithelial Isc, as did substitution of L-Arg with D-Arg or incubation in L-Arg-free medium. Furthermore, the stable and products of Arg metabolism were found to have no effect on epithelial ion transport. These studies show that endotoxin-stimulated alveolar macrophages impair distal lung epithelial ion transport by an L-Arg-dependent mechanism by inactivating amiloride-sensitive 25-pS NSC channels. This may represent a novel mechanism whereby local inflammatory cells regulate lung epithelial ion transport. This could affect the ability of the lung to clear fluid from the air space.