Introduction

Abstract Introduction References

The net flux of liquid into or out of the lung lumen results from a balance of active liquid secretory and absorptive forces. During fetal life, liquid is secreted by the epithelium lining the acini and small airways of the developing lung. Liquid secretion results from the active transport of Cl from the interstitium to the lumen. Around the time of birth, this secreted liquid must be cleared rapidly from the future air spaces to allow adaptation to independent respiration (1). It has long been proposed that Na⁺ absorption through amiloride-sensitive channels is the driving force for the rapid clearance of fetal-lung liquid from the lung at birth (2). The recent cloning of the amiloride-sensitive epithelial Na⁺ channel, ENaC (3), has greatly advanced our understanding of Na⁺ transport in the lung. Definitive evidence that ENaC plays a critical role in perinatal lung liquid clearance came from recent studies of mice that were deficient in the -subunit of ENaC (4). Affected mice were unable to clear liquid from their lungs at birth and died within the first 2 d of life with lung water content that was not different from fetal values.

The precise function of the three individual subunits of ENaC, , and in perinatal and postnatal lung liquid balance has not yet been established. Oocyte expression studies suggest that all three subunits are required for optimal function of channel, and that only the  subunit appears to have intrinsic ion-transporting capacity (3, 5). To explore further the role of individual ENaC subunits in liquid absorption during fetal and postnatal life, we mapped regional murine ENaC (mENaC) subunit messenger RNA (mRNA) expression by in situ hybridization and quantified changes in subunit expression by Northern blot analysis. mENaC mRNA subunit expression was studied from Day 14 through Day 19 of murine fetal lung development (pseudoglandular and cannilicular phases), in the early postnatal period (terminal sac development) and in adult mouse lung.

	Materials and Methods

Animals

Timed pregnant C57/B16 mice were obtained from Jackson Laboratories (Bar Harbor, ME). After maternal CO₂ inhalation, fetuses were dissected free from the uterus and lungs were dissected free from heart and great vessels. For Northern analysis, fetal lungs were pooled from 15-d (four litters, 24 fetuses), 17-d (three litters, 22 fetuses), and 19-d (two litters, six fetuses) mice to obtain sufficient RNA for the analysis. For postnatal time points, four mouse lungs were used for Day 1 analysis, and two lungs or lung segments were used for the remaining time points. For in situ studies, lungs from three fetuses from each gestation (14 to 19 d) and postnatal (1 wk and adult) time point were studied.

Preparation of Mouse-Specific ENaC Probes by Reverse Transcriptase

Total RNA was isolated from mouse lung and flash-frozen in liquid N₂. Reverse transcription of murine lung RNA was primed with a cocktail of three oligonucleotides (specific for each subunit) derived from the 3' end of the rat sequence specific for each subunit (6). The complementary DNA from these reactions was used to amplify specific fragments, via polymerase chain reaction (PCR), of 636 base pairs (bp) (mENaC), 429 bp (mENaC), and 678 bp (mENaC). These fragments were subcloned into the PCR II vector using the Stratagene TA cloning kit (Stratagene, La Jolla, CA). The  fragment has been sequenced and is about 98% homologous to rat ENaC; however, the sequences for the - and mENaC fragments have not been confirmed by sequential analysis.

Northern Blot Hybridization

Whole lungs were excised from fetal (15 to 19 d) and postnatal (1 d, 1 wk, 4 wk, and adult [> 8 wk]) mice and dissociated in guanidine isothiocyanate using a Polytron PT1200 (Brinkmann Instruments Inc., Westbury, NY). RNA was purified from mouse lung as described previously (6). Approximately 10 µg/lane of total RNA was fractionated by electrophoresis on a 1.2% agarose-formaldehyde gel. Total RNA was transferred to a Duralon nylon membrane (Stratagene) by capillary blotting according to manufacturer's protocols. The membrane was ultraviolet-crosslinked and stored at 20°C until hybridization. Membranes were prehybridized at 42°C for 1 h in a 20-mM NaPO₄ buffered solution containing 50% deionized formamide, 5× saline sodium citrate (SSC), 1× Denhardt's, 1% glycine, and 250 µg/ml sonicated salmon-sperm DNA (Sigma Chemical Co., St. Louis, MO). The membranes were then hybridized with ³²P-labeled probes generated from purified DNA fragments, corresponding to either -, -, or mENACs, using the Prime-a-Gene Labeling System (Promega, Madison, WI). Hybridizations were performed in 50% formamide, 5× SSC, 1× Denhardt's, 10% dextran sulfate, and 100 µg/ml salmon-sperm DNA at 42°C for 18 h. The membrane was washed at a final stringency of 0.1× SSC/0.1% sodium dodecyl sulfate at 65°C and exposed to Kodak BioMax X-ray film (Eastman Kodak, Rochester, NY) with intensifying screens. Developmental expression levels of mENAC RNA were quantified by densitometry (Molecular Dynamics, Sunnyvale, CA). mENAC RNA levels were normalized to the 28S ribosomal band (stained by methylene blue) to control for variance in loading and transfer of Northern blots, as described previously (7). The length of mENaC RNA was calculated by plotting the log (length [bp]) of 28S and 18S ribosomal bands versus the migrating distance (cm) of the ribosomal bands. Migrating distances of mENaC subunits were determined from a standard curve.

In Situ Analysis for the ENaC Subunits in Fetal Mouse Lung

Frozen sections (8 µm) were mounted on slides and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h. After fixation, slides were rinsed twice in PBS, dehydrated, air-dried, and stored at 20°C until use. In situ hybridization was performed by standard methods, as described previously (8). Briefly, prehybridization consisted of proteinase K digestion, then acetylation. Serial sections were hybridized overnight at 54°C in a hybridization buffer containing 10⁶ counts/min¹ of either antisense or sense ³⁵S-uridine triphosphate-labeled RNA probes (636, 429, 678) synthesized by in vitro transcription with T7 polymerase (according to manufacturer's instructions). After hybridization, slides were washed in 4× SSC at room temperature and subjected to the following sequential protocol: ribonuclease A digestion (20 mg/ml) for 30 min at 37°C, 2× SSC/1 mM dithiothreitol (DTT) at room temperature, a high-stringency wash of 0.5× SSC/ 1 mM DTT at 58°C (3 × 15 min), followed by ethanol dehydration. Dried slides were dipped in Kodak NTB2 photoemulsion and stored at 4°C until developed. Slides were developed at intervals from 3 to 11 d, counterstained with hematoxylin and eosin (H&E), and photographed using brightfield and darkfield microscopy at ×40 magnification (Nikon Microphot-SA microscope).

	Results

Whole-Lung mENaC Subunit mRNA Expression: Northern Blot Analysis

Northern Blot analysis of mRNA isolated from adult rat whole-lung preparations (Figure 1) showed intense expression of mENaC (1.0 densitometric units) and lesser expression of mENaC (0.26 units) and mENaC (0.40 units). The length for each mENaC subunit was determined: mENaC = 3,700 bp, mENaC = 2,800 bp, mENaC = 3,300 bp. Relative subunit expression during development and postnatal life was expressed as a percentage of the maximal expression for each subunit (Figure 2). Low mRNA levels of all three mENaC subunits were detected at 15 d. All three subunits showed a significant increase in expression late in gestation (i.e., Days 17 to 19). - and mENaC expression levels reached near-adult levels by the first day of postnatal life. Postnatal mENaC levels increased more gradually, progressively increasing through to adult life.

View larger version (48K): [in this window] [in a new window]   Figure 1.   Northern blot analysis of ENaC subunits. Northern blot of adult lung mENaC mRNA showing the relative intensity of expression and size of each subunit. Length of subunits: mENaC, 3,700 bp; mENaC, 2,800 bp; mENaC, 3,300 bp.

View larger version (19K): [in this window] [in a new window]   Figure 2.   Relative intensity of -, -, and mENaC mRNA for whole lung at different fetal gestations and postnatal ages. mRNA expression intensity is relative to maximum expression for each subunit (maximum = 100%). Maximum expression occurred on Day 1 for mENaC, at 1 mo for mENaC, and at 1 wk for mENaC. Values are means ± standard deviation of pooled samples for each fetal gestation and postnatal age (two pooled samples each for  and , and three pooled samples for ).

Regional mENaC Subunit mRNA Expression in Fetal Lung: In Situ Hybridization

mENaC expression. mENaC mRNA expression was not detected in developing lung before 16 d gestation (Figure 3, Table 1). At 16 d, expression was observed in the central bronchi only. At 17 d, mENaC signal was seen in central bronchi with fainter signal in distal airways. At 18 d, signal was seen throughout the airways and developing acinar structures with stronger, more diffuse signal observed in 19-d lung.

View larger version (113K): [in this window] [in a new window]   Figure 3.   In situ hybridization study of mENaC mRNA expression in fetal lung. Brightfield (H&E) image is shown on the left. Darkfield images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each time point. a, small airway; b, blood vessel. Magnification: ×40.

mENaC expression. mENaC mRNA expression was not detected in 14- or 15-d lung (Figure 4, Table 1). Signal appeared at 16 d in the central lobar airway and, more faintly, in the developing peripheral airways. In 17-d lung, strong signal was seen in lobar and small airways, but not in the developing acini of the peripheral lung. Figure 4 shows an abrupt discontinuation of signal at the distal end of the small airways of 17-d lung. At Days 18 and 19, mENaC mRNA signal is observed primarily in the lobar airways, and only faintly in the small airways and developing acinar structures.

View larger version (122K): [in this window] [in a new window]   Figure 4.   In situ hybridization study of mENaC mRNA expression in fetal lung. Brightfield (H&E) image is shown on the left. Darkfield images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each time point. a, small airway; b, blood vessel. Arrow shows the abrupt discontinuation of mENaC signal in the small airway of 17-d fetal lung. Magnification: ×40.

mENaC expression. mENaC mRNA expression was observed from 15 d (Figure 5, Table 1). mRNA was detected initially in lobar airways and primitive distal tubules of 15-d lungs. In 16-d lung, mENaC mRNA expression was observed to track out to the most distal branches of the airway and the primitive acinar unit. In 17-d lung, all visible epithelium in the distal bronchial tree and developing acini was outlined by mENaC mRNA signal. The mENaC signal in the distal airways and developing alveoli was more intense with advancing fetal gestation.

View larger version (124K): [in this window] [in a new window]   Figure 5.   In situ hybridization study of mENaC mRNA expression in fetal lung. Brightfield (H&E) image is shown on the left. Darkfield images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each time point. a, small airway; b, blood vessel. Magnification: ×40.

Regional mENaC Subunit mRNA Expression in Postnatal Lung: In Situ Hybridization

At 1 wk of postnatal age and in adult lung, intense mRNA expression of all three subunits was observed in medium and small airways (Figure 6, Table 1). In 1-wk lung,  and  subunits were expressed in the alveolar region but with less intensity than in airway epithelium. By contrast, similar intensity of mENaC subunit mRNA expression was seen in alveolar and small airway regions of 1-wk lung. In the adult lung, the patchy distribution of  and  subunit expression in the distal lung suggests that these subunits are localized to alveolar type II (ATII) cells. Expression of mENaC subunit mRNA in the alveolar region of adult postnatal lung was weaker than that observed for mENaC and mENaC.

View larger version (108K): [in this window] [in a new window]   Figure 6.   In situ hybridization study of -, -, and mENaC mRNA expression in 1-wk and adult distal lung. Brightfield (H&E) image is shown on the left. Darkfield images of frozen sections exposed to 35S-labeled antisense and sense mRNA probes are shown for each time point. a, airway; ar, arteriole; v, vein. Magnification: ×40.

	Discussion

At this point the role of individual ENaC subunits in amiloride-sensitive Na⁺ transport is not known. The changes in regional localization and whole-lung quantitation of mENaC subunits in the late fetal and perinatal periods can be viewed in the context of known changes in liquid and ionic flow that occur in the lung at this time.

There is now strong evidence that the switch from liquid secretion in the fetal lung to net liquid absorption in the perinatal and postnatal lung is mediated by activation of the amiloride-sensitive Na⁺ channel ENaC (2, 4, 9). The pattern of changes in mENaC subunit expression from fetal to postnatal life as detected by Northern blot analysis in this study is similar to that reported for rat whole lung (10). ENaC, ENaC, and ENaC subunit expression increase exponentially before birth in both species. However, we did not observe the pronounced postnatal decline and secondary increase in mENaC and mENaC expression levels described for rat lung. We also saw a more progressive increase in mENaC subunit expression through fetal and postnatal life than is seen in rat, with most of the increase in mENaC subunit expression occurring after birth, between 4 and 8 wk of postnatal life. In human lung, ENaC mRNA levels also increase sharply between fetal and postnatal life (11), consistent with our observations in mouse lung.

The changes in ENaC subunit expression are associated with no change in fetal rat lung liquid volume (12) and, at most, a modest slowing of basal secretion rate in fetal sheep (13, 14). However, the capacity for amiloride-sensitive liquid absorption in late-gestation fetal sheep lung can be revealed by -adrenergic stimulation (13). This cyclic adenosine monophosphate (cAMP)-sensitive Na⁺ transport capacity is developmentally regulated and is very likely to be linked to an increase in ENaC subunit expression, as was observed in the present study. The speed with which this switch from secretion to absorption occurs (minutes) is likely to be too rapid for new ENaC synthesis. These observations suggest that ENaC is preassembled in fetal lung but remains inactive until phosphorylated by a rise in cAMP associated with the perinatal surge in fetal epinephrine.

The significance of the variation in timing and localization of the three mENaC subunits as detected by in situ hybridization studies is difficult to interpret without better understanding of the interactions between ENaC subunits and the stochiometry of the subunits in channel assembly. In the oocyte expression system, - and ENaC subunits show no Na⁺ currents when expressed alone or together (3, 5). ENaC has a trivial intrinsic Na⁺ transport capacity when expressed alone, but Na⁺ transport is augmented when this subunit is coexpressed with - or ENaC subunits. However, Na⁺ currents are an order of magnitude greater when all three subunits are coexpressed in this system.

The coexpression of ENaC and ENaC subunits in fetal and postnatal lung, particularly in the alveolar region, may modulate liquid flow across the epithelium. The presence of only two of the three subunits may account for the modest effects of liquid flow of these mENaC subunits in late-gestation fetal lung. These subunits appear to be expressed diffusely in fetal lung, whereas expression is patchy in adult lung. The uniform morphology of late-gestation acinar epithelium makes it difficult to determine whether or not mENaC subunit expression is cell-specific. However, high-power examination of the fetal lung exposed to mENaC or mENaC antisense shows that silver grains are localized to only about 50% of the cells (data not shown). It is possible that these mENaC subunits may be localized to primitive ATII cells, which are more abundant in fetal than in postnatal rodent lung (15). The coexpression of all three subunits at high levels of expression in the small airways suggests that this is the site of maximal liquid absorption in both perinatal and postnatal lung. The perinatal changes and tissue localization of mENaC subunits closely parallels that of Na/K adenosine triphosphatase subunits (16, 17), suggesting a mechanism for coordinated absorption of liquid in specific regions of the perinatal and adult lung.

The distribution of mRNA expression in the postnatal lung is similar to that reported for rat and human species. In rat lung, all three subunits are intensely expressed in the epithelia of small and medium-sized airways (18). In addition, ENaC is expressed in the alveolar region with a punctate distribution that is very similar to surfactant protein C mRNA localization, suggesting that ENAC mRNA is localized in this region to ATII cells (19). Similar localization of all three ENaC subunits to epithelium of human bronchial and nasal airway was described by Burch and coworkers (6). However, expression of ENaC subunit in human lung was less intense than that observed in mouse lung.

We showed previously that exposure of the epithelium to oxygen decreases liquid production by the fetal distal lung epithelium, suggesting that the switch from relative hypoxia during fetal life to normoxia at birth is another trigger that initiates liquid absorption at birth (12). Amiloride-sensitive short-circuit current and ENaC mRNA expression (all three subunits) increases after transfer of fetal rat lung distal epithelial cells from low (fetal) to normoxic (postnatal) culture conditions (20). Our observations suggest that most of the ENaC subunits are in place and ready for the rapid clearance of lung liquid that occurs in the first few hours of life. The effect of the switch in ambient O₂ on subunit expression is probably limited because only modest increases in mENaC and mENaC expression are seen after birth. Patch-clamp studies suggest that some of the effect of O₂ may result from a more direct effect on ENaC channel function (21).

The combination of Northern blot analysis of whole lung and in situ hybridization studies has allowed a more detailed interpretation of the complex maturational and regional changes in Na⁺ transport that occur in the mammalian lung, and provides a framework for the study of factors that regulate ENaC subunits and Na⁺ transport in fetal, perinatal, and postnatal life. Our data suggest that ENaC regulates liquid absorption by quantitative and regional changes in subunit expression, and by selective association of individual ENaC subunits in different regions of the lung.