Developmental regulation of epithelial sodium channel subunit mRNA expression in rat colon and lung

Shigeru Watanabe1, Kazumichi Matsushita1, John B. Stokes1, and Paul B. McCray Jr.2

Departments of 1 Internal Medicine and 2 Pediatrics, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Na+ absorption via amiloride-sensitive Na+ channels is of critical importance in the transition between fetal and neonatal life in several tissues, including the colon, lung, and kidney. To characterize and contrast the mRNA expression of each of the three epithelial Na+ channel complex (ENaC) subunits, we conducted RNase protection assays (RPA) and in situ hybridization in colon and lung in fetal (17, 19, 20, and 21 days) and postnatal (1, 3, 9, 15, and 30 days) rats (r). In the colon the alpha -, beta -, and gamma -rENaC subunits showed quantitatively different but qualitatively similar expression. All three subunits gradually increased in abundance from fetal day 19 through day 30 of life. The amount of each subunit on day 30 was approximately three times the amount at day 1. In situ hybridization showed that each subunit was localized to the surface epithelial cells with minimal expression in the crypts. The lung showed a completely different pattern. In contrast to the colon, the total amount of alpha -rENaC mRNA (by RPA) in the lung increased dramatically from fetal day 19 to 21, whereas beta - and gamma -rENaC showed modest prenatal increases. The amounts of all three mRNAs fell after birth through day 9 (to about 75% of the day 1 value). On days 15 and 30 the amount of mRNA rose to approach the values on day 1. alpha -rENaC mRNA abundance always exceeded beta - and gamma -rENaC, and the quantitative expression was different for alpha - than for beta - and gamma -rENaC. In situ hybridization studies showed that all three subunits were expressed in epithelial cells of the bronchi, bronchioles, and alveoli and not in blood vessels. These studies show striking developmental heterogeneity in rENaC mRNA expression between lung and colon, probably reflecting different developmental regulatory mechanisms in these organs.

glucocorticoid; mineralocorticoid; epithelial sodium channel complex

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ELECTROGENIC TRANSPORT of Na+ occurs across many epithelial tissues, including the colon, lung, and kidney. This process, which involves the translocation of Na+ across cell membranes through specific ion channels, plays an important role in the absorption of fluid. The ability to absorb Na+ through epithelial channels is of critical importance in the transition between fetal and neonatal life. In the colon and kidney, Na+ absorption is critically important for the regulation of fluid and electrolyte homeostasis and nutritional status during the early postnatal period. In the fetal lung, net secretion of (Na)Cl and fluid is replaced by net absorption of Na(Cl) and fluid at the time of birth, a process essential for perinatal adaptation (44). This absorptive process is inhibited by amiloride, suggesting that it takes place through amiloride-sensitive Na+ channels (34, 44). The molecular pathway believed to be responsible for this Na+ transport is the epithelial Na+ channel complex (ENaC). A striking example of the role of ENaC is seen in genetically engineered mice lacking the alpha -subunit; these animals die within 48 h of birth from failure to clear fetal lung fluid (20).

Electrogenic Na+ transport by the colon occurs via ENaC, and it is regulated in the postnatal period (38). Although the genes encoding the alpha -, beta -, and gamma -subunits of rat (r) ENaC were originally cloned from colon (9, 10), less is known about the process of developmental regulation of ENaC in the colon than in the lung. Because the epithelial Na+ channel encoded by rENaC plays a significant role in fluid transport in the colon and lung, we hypothesized that the mRNA expression pattern during the transition from fetal to postnatal life would reflect the roles they play during this period. To understand the ontogeny of the rENaC subunit expression and factors that may regulate expression we determined steady-state mRNA expression in rat colon and lung and localized the mRNAs using in situ hybridization.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue Preparation

Timed pregnant (sperm positive day 0) Sprague-Dawley rats were obtained from Sasco (Omaha, NE). Maternal animals were anesthetized with methoxyfluorane, exsanguinated, and the fetuses were removed and placed in ice-cold Hank's solution. Neonatal rats were also anesthetized with methoxyfluorane. Fetal lung and descending colon tissues were obtained from days 17, 19, 20, and 21 of gestation and postnatal days 1, 3, 9, 15, and 30. Fetal tissues were pooled for analysis as follows: fetal day 17 and 19, four lung specimens, six colon specimens; fetal day 20 and 21, two lung specimens, four colon specimens; postnatal day 1, one lung specimen, two colon specimens; all other time points, one specimen per analysis. Pooled samples were used only for a single measurement. The protocol was approved by the Animal Care and Use Committee of the University of Iowa.

Preparation of RNA

The descending colon was rinsed with ice-cold PBS, and the most distal tissue was trimmed and frozen. The lungs were rapidly removed and immersed directly in liquid nitrogen. Total RNA was isolated from tissues using a modification of the method of Chomczynski and Sacchi (12). An amount of tissue estimated to yield ~100 µg total RNA was homogenized using a 92/Polytron PT-DA 3012/2 TS probe in 2.0 ml of TRI reagent (Molecular Research Center, Cincinnati, OH) for 3-5 s, and the RNA was extracted in 0.2 ml chloroform. After precipitation with isopropanol, the RNA was rinsed with 75% ethanol, centrifuged for 5 min at 7,500 g, air dried for 5-10 min, and resuspended in diethyl pyrocarbonate-treated water. Total RNA was measured by absorption spectrophotometry at 260 nm. The 260/280 ratio averaged 1.6-1.8.

RNase Protection Assay

The plasmids used to make the antisense probes for the RNase protection assays (RPA) were constructed as previously described for alpha -, beta -, and gamma -rENaC (27, 43). The alpha -rENaC construct was a 422 nt segment corresponding to nucleotides 223-645 of the coding region (9) subcloned into the pKS(-) vector (Stratagene, La Jolla, CA) using restriction sites Eco RI and Pst I. The beta -rENaC construct was a 249 nt segment subcloned into the vector pCR-Script SK(+) (Stratagene) using restriction sites Pst I and Sac I. The gamma -rENaC construct was a 675 nt segment subcloned into the pCRII-TA vector (Invitrogen, Carlsbad, CA) using restriction sites Xba I and Bst XI. All of the probes used for rENaC subunits were directed against unique segments within the open reading frame.

Antisense probes for the RPA were synthesized from the appropriate constructs using the BrightStar BIOTINscript nonisotopic in vitro transcription kit (Ambion, Austin, TX). The amount of biotin-labeled CTP was adjusted to give the highest possible specific activity. The lengths of the biotin-labeled, unprotected fragments were 480, 280, and 750 nt for alpha -, beta -, and gamma -rENaC, respectively.

We used an 18S antisense probe to normalize for the amount of RNA assayed. This denominator probe was selected over the more traditional actin or glyceraldehyde-3-phosphate dehydrogenase because these latter probes might be developmentally regulated in an organ-specific fashion, thus making comparisons difficult. The human 18S cDNA (obtained from Dr. Jean Robillard) consisted of an unprotected fragment of 110 nt. Because the abundance of the 18S mRNA is several orders of magnitude greater than that of the rENaC subunits, we constructed separate biotin-labeled and unlabeled antisense RNA probes. Based on extensive preliminary testing, we determined that a mixture of 1:1,000 (labeled:unlabeled, total of 1 µg antisense RNA) saturated the 18S mRNA in the reaction and provided a signal that was easily detectable under our experimental conditions. The calculated molar ratio of 18S probe to 18S mRNA was 5:1. This value agreed well with experimentally determined ratios.

The hybridization of ~1 ng of each of the rENaC probes and 1 µg of 18S probe with 25 µg total RNA from each of the tissues was conducted using the RPAII ribonuclease protection assay kit (Ambion). Hybridization was conducted overnight at 45°C. Digestion with RNase A and RNase T1 (at a final concentration of 250 and 10,000 U/l, respectively) was conducted at 37°C for 30 min. The products were subjected to electrophoresis through a 5% denaturing polyacrylamide-8 M urea gel buffered with Tris borate for 2 h at 250 V and transferred to a nylon membrane (BrightStar Plus, Ambion) using a semidry electroblotter (Fisher, Itasca, IL). The membrane was subsequently ultraviolet (UV) cross-linked (UV Stratalinker, Stratagene), and development of the protected RNA fragments was conducted using the BrightStar Biodetect nonisotopic detection kit (Ambion) with minor modifications. To reduce background noise the wash times after incubation with the streptavidin-alkaline phosphatase conjugate solution were increased threefold. The developed blots were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) for 1-45 min, depending on the intensity of the product.

The gamma - and alpha -rENaC probes and the 18S probe occasionally showed degradation products when the amount of the respective mRNA was large. The magnitude of these products was <10% of the completely protected fragment, and the shorter fragments did not interfere with the ability to quantitate any of the other bands of interest. Therefore quantitation of these transcripts was conducted on the major protected fragments. The autoradiograms were quantitated with a PDI scanning densitometer using Quantity One software (Huntington Station, NY). The exposure time of the autoradiograms was adjusted so that the density of the majority of the bands on a given gel fell into the linear range of the instrument.

In Situ Hybridization

Colon and lung samples were prepared from four animals from each time point (2 for paraffin, 2 for frozen section) for use in in situ hybridization as previously reported (27). We used both frozen and paraffin sections for all probes, recognizing that there might be some advantages to one form of fixation over the other. Tissues were immersed in 2% paraformaldehyde in PBS for 2 h at 4°C, embedded in paraffin, sectioned to 5-mm thickness, and mounted onto slides (Superfrost Plus, Fisher Scientific, Fair Lawn, NJ), which were air dried and stored with desiccant at room temperature in an airtight box until they were used for hybridization. Postnatal lung tissues for frozen section were rapidly removed and inflated via trachea with 5 ml of OCT compound (Miles, Elkhart, IN) diluted 1:1 with PBS and frozen in liquid isopentane. Frozen tissues were sectioned on a cryostat to 7-mm thickness and thaw-mounted onto slides. After air drying for a few minutes, slides containing sections were fixed in freshly prepared 4% paraformaldehyde in PBS, pH 7.4, at 4°C for 10 min, dehydrated in graded ethanols, and stored with desiccant at -20°C in an airtight box.

Hybridization was performed on triplicate sections from the same tissue for each of three exposures (4, 8, and 13 wk). Tissues from different animals were used for paraffin-embedded and frozen sections. The prehybridization and hybridization steps were conducted according to standard methods (50) as modified by this laboratory (27). After hybridization, sections were washed at high stringency [50% formamide, 2× saline-sodium citrate (SSC), and 25 mM dithiothreitol] at 60°C for 30 min, rinsed with 4 M NaCl, 10 mM Tris, 5 mM EDTA, and 25 mM dithiothreitol (pH 8), and treated with 20 mg/ml RNase A and 1 U/ml RNase T1 for 30 min at 37°C. After a second series of high-stringency washes and dehydration, the sections were then coated with NTB-2 autoradiography emulsion (Eastman Kodak, Rochester, NY), diluted 1:1 in distilled H2O, air dried, and exposed at 4°C before being developed. Sections were counterstained with toluidine blue.

Probe Preparation

The alpha -rENaC cDNA used for the in situ hybridization studies was the same as that used for the RPA previously described. The beta -rENaC cDNA was a modification of a previously described construct (48) cloned into pCRII-TA vector (Invitrogen). The probe was restricted with Spe I to remove the 3'-poly A tail and religated. The plasmids containing the alpha - and beta -rENaC probes were linearized with the appropriate restriction enzymes, and sense (T3) or antisense (T7) labeled probes were prepared using the Riboprobe Gemini II Core System (Promega, Madison, WI) transcription kit substituting 11 mM 35S-UTP (Amersham, Arlington Heights, IL) for cold nucleotide. The gamma -rENaC cDNA consisted of the first 567 nt segment of the open reading frame (48) subcloned into pCRII-TA cloning vector (Invitrogen, San Diego, CA). Purified plasmids were linearized with the appropriate restriction enzyme, and sense (T7) or antisense (SP6) 35S-UTP,35S-CTP (Amersham) double-labeled probes were prepared. All in situ hybridization reactions were carried out using serial sections for sense and antisense probes.

Statistical Analysis

All values determined by RPA were normalized to the 18S band (exposed for a shorter period to account for the generally larger signal). In general, the 18S bands were exposed for 1-3 min and all other bands for 15 min. Significance was determined by two-way analysis of variance comparing time (development) effects and subunit differences or organ differences as indicated. P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

rENaC mRNA Expression Is Regulated in Tissue-Specific Manner

Colon. To characterize and quantitate the mRNA expression of each of the three rENaC subunits, we conducted RPA in colon and lung in fetal and postnatal rats. The developmental time course of rENaC mRNA expression in the colon and lung were completely different. The features that characterize colon rENaC expression during development are shown in Figs. 1 and 2. Figure 1A shows an example of an RPA for all three rENaC subunits in colon tissue during development. All three subunits showed a gradual increase in abundance throughout the period examined. The quantitative assessment of the amount of each of the subunits relative to the abundance of alpha -rENaC on day 1 is shown in Fig. 2A. alpha -rENaC appeared to have the greatest abundance throughout development. The amount of mRNA for each subunit was greater on day 30 than on day 1.


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Fig. 1.   Representative RNase protection assays for alpha -, beta -, and gamma -rENaC in colon (A) and lung (B). A: ontogeny of mRNA expression in rat descending colon. Tissues were taken from fetal (days 17f, 19f, 20f, 21f) and postnatal (days 1, 3, 9, 15, 30) rats. Right lane (B) shows unprotected (UP) riboprobe fragments for gamma -, alpha - and beta -rENaC and 18S ribosomal subunit, respectively. B: ontogeny of mRNA expression in rat lung.


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Fig. 2.   Ontogeny of rENaC mRNA expression in descending colon. All data were normalized to 18S expression. A: values were normalized to level of alpha -rENaC at day 1. * alpha  values different from beta  and gamma  (P < 0.0005). + beta  values different from alpha  and gamma  (P < 0.0005). B: same data as A expressed as ratio at day 1 for each subunit. All 3 rENaC mRNAs show significant increases in expression with time (P < 0.0001). Values represent means ± SE (n = 4 at each time point).

To analyze the relative developmental pattern (i.e., ignoring differences in the amount of mRNA between subunits) we expressed each subunit according to the relative amount on day 1. As shown in Fig. 2B, this analysis demonstrated that the expression pattern for all three subunits over time in the colon was qualitatively similar. The abundance (relative to itself) of alpha -, beta -, and gamma -rENaC in the colon is similar to the amount of each subunit on day 30 being about three times the amount on day 1.

Lung. The lung showed a completely different developmental time course of rENaC mRNA expression from the colon. Figure 1B shows an example of an RPA for all three rENaC subunits in lung tissue during development. Figure 3A shows the analysis of the amount of each of the subunits relative to the abundance of alpha -rENaC on day 1. This analysis indicates that the amount of alpha -rENaC is several-fold greater than beta - or gamma -rENaC at all time points after fetal day 19. It also shows the biphasic nature of alpha -rENaC mRNA expression with peaks around the time of birth and again 2-4 wk later.


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Fig. 3.   Ontogeny of rENaC mRNA expression in lung. All data were normalized to 18S expression. A: values were normalized to day 1 alpha -rENaC (set at unity). All 3 subunits showed prenatal rise in expression (P < 0.05), although increase was greater with alpha -rENaC (*) than with beta - and gamma -subunits (P < 0.0005). B: same data as A expressed as ratio at day 1 for each subunit. Values represent means ± SE (n = 4 at each time point). Relative expression is different for alpha - than for beta - or gamma -subunits (* P < 0.05). All 3 rENaC mRNAs show significant increases in expression with time (P < 0.0001).

To analyze the relative developmental pattern (i.e., ignoring differences in the amount of mRNA between subunits), we again expressed each subunit according to its own abundance on day 1. As shown in Fig. 3B all three subunits showed at least a tendency to the biphasic response noted with alpha -rENaC. Note that for each subunit the amount of mRNA on day 30 is not greater than on day 1. This analysis also shows a significant difference in the pattern of expression between alpha -rENaC and the other two subunits. The clearest difference is the rate of increase between fetal days 19 and 21 when the rate of increase of beta - and gamma -rENaC were identical but slower than the increase in alpha -rENaC. After birth the relative amount of each rENaC subunit fell, and the quantitative correlation between beta - and gamma -rENaC became somewhat looser. However, this apparent dissociation is magnified by this particular analysis; the data in Fig. 3A show that beta - and gamma -rENaC track much more closely with each other than they do with alpha -rENaC. Together, these two analyses demonstrate three features: 1) after fetal day 19, alpha -rENaC mRNA was always more abundant than beta - and gamma -rENaC, 2) the rate of increase in alpha -rENaC mRNA before birth was greater with alpha - than with beta - or gamma -rENaC, and 3) the qualitative pattern of expression was similar for all three subunits.

Figure 4 compares the pattern of expression for each of the three subunits in colon and lung. The major difference for each subunit is that the relative abundance in the colon increases after day 1, whereas the abundance in the lung peaks at day 1.


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Fig. 4.   Ontogeny of ENaC mRNA expression in lung and descending colon. Data for each subunit were normalized to level of expression on day 1. Each subunit has different pattern in lung and colon (* P < 0.0005 for alpha , beta ; P < 0.001 for gamma , beta ). A: alpha . B: beta . C: gamma .

rENaC mRNA Expression Is Epithelial Cell Specific in Developing Colon and Lung

We used in situ hybridization to localize rENaC subunit mRNA expression. Localization was detectable in the early postnatal period in both the colon and lung. The in situ hybridization pattern in the colon was quantitatively similar to what we might have expected from the RPA analysis. We were unable to detect any signal before postnatal day 9. By day 15, expression of each subunit was localized to the surface epithelial cells with little signal in the crypts (Fig. 5, A-F).


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Fig. 5.   Localization of rENaC mRNA in descending colon by in situ hybridization on day 15 of life. A, C, and E: brightfield photomicrographs of darkfield areas shown in B, D, and F, respectively. Expression of alpha -, beta -, and gamma -rENaC is seen over surface epithelia of colon (arrows in B, D, and F). Control sense riboprobes detect no specific signal (not shown).

Representative photographs of the in situ hybridization results are shown in Fig. 6. All three subunits were expressed in epithelial cells of the bronchi and bronchioles during the perinatal period. No expression was detected in blood vessels. alpha -rENaC was evident in alveolar epithelia at postnatal day 1, whereas beta - and gamma -rENaC were undetectable in alveoli (Fig. 6, B, E, and H). The pattern on postnatal day 15 was similar; however, at this point both beta - and gamma -rENaC mRNAs were detected in alveolar epithelia (Fig. 7, B, E, and H). The expression pattern in the alveoli was similar to that seen in the adult rat lung and suggests expression in type II pneumocytes (16, 27).


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Fig. 6.   Localization of rENaC mRNA in lung by in situ hybridization on postnatal day 1. Expression of all 3 subunits was detected in bronchioles (B). A, D, and G: brightfield photomicrographs of darkfield areas shown in B, E, and H, respectively. Sense probes (C, F, and I) analyzed on adjacent sections showed no specific signal. alpha -rENaC expression (A-C) but not beta - (D-F) or gamma -ENaC (G-I) is detected in alveolar epithelia. Arrows show alveoli. Blood vessels (V) are negative.


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Fig. 7.   Localization of rENaC mRNA in lung by in situ hybridization on postnatal day 15. Expression of all 3 subunits is evident in bronchi (Br) and bronchioles (B) and in alveolar epithelia. Control sense riboprobes detect no specific signal (C, F, I). Arrows in B and H show alveoli.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present studies show marked developmental heterogeneity in rENaC mRNA expression between colon and lung. In the colon prenatal increases in rENaC mRNA expression in the descending colon are modest, but a significant postnatal rise occurs between days 3 and 30. In contrast, lung rENaC mRNA upregulation begins after fetal day 19 with peak expression by the first postnatal day. The cellular localization patterns for rENaC mRNA in colon and lung are similar to that reported in adult rat tissues (15, 27). These results show that rENaC developmental expression is regulated in a temporal and tissue-specific manner, reflecting different regulatory mechanisms in these organs.

There are few data on rENaC expression in the colon during development. Expression of alpha -rENaC mRNA increases during development (14, 49), but the ontogeny of the beta - and gamma -rENaC subunits in the colon has not been described. A recent report identified a developmental aspect that we did not detect (14). A truncated (1.2 kb) alpha -ENaC transcript missing the first ~800 nt of the coding region was noted in mouse colon transiently before birth (14). Because our riboprobe hybridizes to this region, we would not have detected this shorter transcript. However, it is not likely that this shorter transcript would have a similar function as the full length because it would be predicted to be missing the first membrane spanning domain.

Aspects of the ontogeny of ENaC subunit mRNA expression in the lung have been reported in the rat (14, 33, 45) and human (29, 30, 47). Our results are similar to those of O'Brodovich and co-workers (33, 45) who showed a biphasic expression of rat alpha -ENaC with peaks around the time of birth and again in adult life. The timing of the second increase was not reported in their work. Dagenais and colleagues (14) also noted a perinatal increase in alpha -ENaC mRNA expression in developing mouse lung but had limited postnatal time points.

However, our results differ from those previously reported for the pattern of beta - and gamma -rENaC expression in the lung. Whereas the previous report (45) did not note a biphasic response of these subunits, we can clearly detect this response (Fig. 3B). The reason may be due to the differences in rat strain or to the nature of the assay. We suspect that the latter explanation is correct. Ribonuclease protection assays are 10- to 100-fold more sensitive in detecting low abundance mRNAs than Northern hybridization. Thus our study might have detected more subtle changes in the expression of the lower abundance beta - and gamma -rENaC mRNAs in the lung.

Functional Importance of ENaC in Development

The epithelial-specific expression patterns of the rENaC subunits in the colon and lung are consistent with the ENaC complex being important in mediating developmentally regulated electrogenic Na+ absorption in these organs. Amiloride-sensitive Na+ absorption across the rat descending colon begins at the end of the first week of life and increases through the weaning period (38). The gradual increase in ENaC subunit mRNA expression reported here (Fig. 2) correlates well with the presumed functional consequences (Na+ transport). Studies in humans suggest that colonic salt absorption is present by late in the third trimester and that this route of Na+ conservation may be more important than the kidney in the neonatal period, especially in the preterm infant (1, 21). Amiloride-sensitive current is also present in the surface epithelium of the descending colon where rENaC mRNA is expressed in the neonate (this study) and adult (15, 40). Thus colonic Na+ absorption via ENaC may be an important nutritional source of Na+ to support the rapid growth that takes place in the neonatal period (17).

Although there are few studies of ion transport across rat airways in vivo or in vitro (7, 23), amiloride-sensitive short-circuit current has been reported in the rat trachea (23). In other species amiloride-sensitive Na+ transport is well documented throughout the pulmonary epithelium. For example, studies of the ion transport pathways across airway epithelia of human (5, 6, 22) and mouse airways document the presence of significant amiloride-sensitive short-circuit current. Amiloride-sensitive Na+ transport also accounts for the majority of the short-circuit current present in monolayer cultures of alveolar type II cells from adult rats (13, 26). Measurements of the bioelectric properties of distal lung epithelia from fetal rats (35, 39) or humans (3, 28) also show evidence of significant amiloride-sensitive apical membrane Na+ current. Thus these in situ studies support the notion that rENaC is responsible for electrogenic Na+ transport in colonic and pulmonary epithelia.

Factors Potentially Responsible for ENaC Expression

The contrast in the onset of rENaC mRNA expression between the colon and lung implies that different regulatory factors are involved. There are several developmental aspects that might explain these differences. For purposes of simplicity, we will discuss the early (prenatal) response separately from the later (postnatal) response.

We consider the prenatal increase in alpha -rENaC mRNA to be caused by the prenatal surge of corticosterone. Although this hypothesis has yet to be proven conclusively, there are two lines of evidence that strongly support it. First, the increase in fetal corticosterone occurs immediately before the increase in the abundance of alpha -, beta -, and gamma -rENaC (4, 25, and Fig. 3), indicating a strong temporal correlation. Second, synthetic glucocorticoid given before the normal glucocorticoid surge causes an increase in alpha -rENaC mRNA in fetal rat lung (33, 45). Furthermore, cultures prepared from rat fetal lung epithelia (11, 45) or tissue explants (29, 46) show a similar response to synthetic glucocorticoids. Thyroid hormone might also influence ENaC expression in fetal lung, although the evidence for this is conflicting (11, 46).

If the fetal corticosteroid surge increases rENaC mRNA in the lung, why does it not do so in the colon? There are two possible explanations. First, there may be an inadequately developed response mechanism in the colon. For example, exogenous dexamethasone must be given after fetal day 17 for it to produce its effect on alpha -rENaC mRNA in the lung. If given earlier (i.e., before the canalicular stage), dexamethasone apparently has no effect (45). Thus there seems to be a combination of events necessary to effect the increase in alpha -rENaC mRNA in lung. A similar scenario might also occur in the colon, but the events enabling colonic glucocorticoid responsiveness might not be activated by fetal day 19. A second possibility derives from our understanding of organ responsiveness to corticosterone and aldosterone. Both steroid hormones bind to and activate the mineralocorticoid receptor. However, in classic aldosterone-responsive target tissues, such as kidney collecting duct and colon, corticosterone (and cortisol) is inactivated by type II 11-beta -hydroxysteroid dehydrogenase (8, 31). It is possible that the differential effect of corticosterone on lung might be owing to the presence of this metabolizing enzyme in colon as early as fetal day 18. One study presents evidence of type II 11-beta -hydroxysteroid dehydrogenase activity in midgestation human fetal lung and colon (42), whereas another demonstrates conversion of cortisol to cortisone by first and second trimester human fetal lung (32), raising the possibility that the activity of this enzyme influences glucocorticoid responsiveness in fetal tissues.

After the peak expression of rENaC mRNA in the lung around the time of birth, the levels for all three subunits decline. After postnatal day 9 they begin to rise again. These later changes parallel the increases in circulating corticosterone in neonatal life (19). The fall in circulating corticosterone levels in newborn rats probably reflects the switch from predominantly maternal corticosterone production to that of the newborn. During the first 1-2 wk of life rodents are relatively unresponsive to stimuli that increase corticosterone in adults, a period some call the "stress hyporesponsive period" (25, 41). This second parallel increase in lung rENaC mRNA and circulating corticosterone adds to the evidence that this adrenal steroid is a major regulator of lung rENaC. In support of this notion, studies in adrenalectomized adult rats also show that the mRNAs for alpha -, beta - and gamma -ENaC in the lung increase in response to dexamethasone (40).

The mechanism responsible for the increase in rENaC mRNA in the colon after birth is not entirely clear. One possibility is that an increase in circulating aldosterone produces this effect. It is well established that aldosterone levels increase after the first week of life in the rat (37). In addition, there is a strong correlation between the aldosterone levels and the magnitude of amiloride-sensitive short-circuit current across the colon, a measure of ENaC-mediated Na+ transport (37, 38). The temporal relationships between aldosterone levels, the effector mRNA, and the functional effects provide good circumstantial evidence for this explanation. In addition, exogenous aldosterone increases amiloride-sensitive short-circuit current in colons from adult rats (18) and aldosterone administered to 10-day-old rats produces a modest increase in colonic alpha -rENaC mRNA levels (49). From this analysis, one might presume that whereas corticosterone is a major regulator of rENaC expression in lung, aldosterone is the major regulator of rENaC in the colon.

Despite the attractiveness of this scenario, there are two lines of evidence that raise important questions regarding its adequacy. First, administering synthetic glucocorticoid to 10-day-old rats did not increase alpha -rENaC mRNA levels in colon (49). One might explain this lack of a glucocorticoid effect as the result of differential effects of glucocorticoid and mineralocorticoid hormones; they do interact with different receptors. However, it would be the first example of a tissue that responded to mineralocorticoid that did not respond similarly to synthetic (i.e., nonmetabolizable) glucocorticoids. Furthermore, in adult rats dexamethasone increases colonic rENaC mRNA in a pattern similar to that of aldosterone (2, 40). Second, it is now clear that either aldosterone or dexamethasone produces an increase in beta - and gamma -rENaC in adult rat colon without any change in alpha -rENaC mRNA (2, 24, 40). If such steroid effects play a role in the increase of beta - and gamma -rENaC mRNA during development, what causes the increase in alpha -rENaC mRNA in the postnatal period (Fig. 2)? If aldosterone is producing these postnatal effects, it must be doing it via a mechanism that is not currently recognized or that is lost in adults. Clearly, further study is necessary to determine the specific mechanisms responsible for the regulation of these mRNAs and the relationship between the expression of ENaC subunit mRNAs and proteins.

That the ontogeny of rENaC mRNA expression is discordant in colon and lung is consistent with the needs of the organism at specific developmental time points. The increase in postnatal Na+ absorption by the colon is thought to be an important source of nutritional Na+ during a time of rapid growth (1, 21, 38). Upregulation of rENaC in the lung before birth allows the absorption of Na+, which drives the absorption of lung fluid at the time of birth. The absorption of Na+ in the lung via ENaC is essential for survival (20, 34-36).

Because both the ability to absorb Na+ from the colon and the clearance of fetal lung fluid are important for perinatal survival and growth, it is likely that abnormalities in the regulation of ENaC expression might be relevant to disease in premature infants. Colonic and pulmonary Na+ transport may be functionally immature in preterm infants born before the signals regulating ENaC expression are normally active. Thus immaturity of Na+ transport via ENaC may contribute to the pulmonary, electrolyte balance, and nutritional problems of the preterm infant.

    ACKNOWLEDGEMENTS

We thank Monica Driscoll for the alpha -rENaC and gamma -rENaC cDNA clones. We thank Rita Sigmund for technical assistance.

    FOOTNOTES

This work was supported in part by the National Institutes of Health Grant HL-02767 and the Children's Miracle Network Telethon (P. B. McCray) and the National Institutes of Health Grant HL-55006 (J. B. Stokes), the O'Brien Kidney Disease Center National Institutes of Health Grant DK-52617 (J. B. Stokes and P. B. McCray) and a merit award from the Department of Veterans Affairs (J. B. Stokes). S. Watanabe was supported in part by a fellowship from the Iowa Affiliate of the American Heart Association.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: P. B. McCray, Jr., Dept. of Pediatrics, Univ. of Iowa College of Medicine, Iowa City, IA 52242.

Received 23 March 1998; accepted in final form 12 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Al-Dahhan, J., G. B. Haycock, C. Chantler, and L. Stimmler. Sodium homeostasis in term and preterm neonates. II. Gastrointestinal aspects. Arch. Dis. Child. 58: 343-345, 1983[Abstract].

2.   Asher, C., H. Wald, B. C. Rossier, and H. Garty. Aldosterone-induced increase in the abundance of Na+ channel subunits. Am. J. Physiol. 271 (Cell Physiol. 40): C605-C611, 1996[Abstract/Free Full Text].

3.   Barker, P. M., R. C. Boucher, and J. R. Yankaskas. Bioelectric properties of cultured monolayers from epithelium of distal human fetal lung. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L270-L277, 1995[Abstract/Free Full Text].

4.   Barlow, S. M., P. J. Morrison, and F. M. Sullivan. Plasma corticosterone levels during pregnancy in the mouse: the relative contributions of the adrenal glands and foeto-placental units. J. Endocrinol. 60: 473-483, 1974[Medline].

5.   Boucher, R. C. Human airway ion transport. Part I. Am. J. Respir. Crit. Care Med. 150: 271-281, 1994[Medline].

6.   Boucher, R. C. Human airway ion transport. Part II. Am. J. Respir. Crit. Care Med. 150: 581-593, 1994[Medline].

7.   Boucher, R. C., Jr., P. A. Bromberg, and J. T. Gatzy. Airway transepithelial electric potential in vivo: species and regional differences. J. Appl. Physiol. 48: 169-176, 1980[Abstract/Free Full Text].

8.   Brem, A. S., and D. J. Morris. Interactions between glucocorticoids and mineralocorticoids in the regulation of renal electrolyte transport. Mol. Cell. Endocrinol. 97: C1-C5, 1993[Medline].

9.   Canessa, C. M., J.-D. Horisberger, and B. C. Rossier. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[Medline].

10.   Canessa, C. M., L. Schild, G. Buell, B. Thorens, I. Gautschi, J.-D. Horisberger, and B. C. Rossier. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[Medline].

11.   Champigny, G., N. Voilley, E. Lingueglia, V. Friend, P. Barbry, and M. Lazdunski. Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones. EMBO J. 13: 2177-2181, 1994[Abstract].

12.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

13.   Cott, G. R., K. Sugahara, and R. J. Mason. Stimulation of net active ion transport across alveolar type II cell monolayers. Am. J. Physiol. 250 (Cell Physiol. 19): C222-C227, 1986[Abstract/Free Full Text].

14.   Dagenais, A., R. Kothary, and Y. Berthiaume. The alpha  subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression. Pediatr. Res. 42: 327-334, 1997[Abstract].

15.   Duc, C., N. Farman, C. M. Canessa, J. Bonvalet, and B. C. Rossier. Cell-specific expression of epithelial sodium channel alpha , beta , and gamma  subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J. Cell Biol. 127: 1907-1921, 1994[Abstract].

16.   Farman, N., C. R. Talbot, R. Boucher, M. Fay, C. Canessa, B. Rossier, and J. P. Bonvalet. Noncoordinated expression of alpha -, beta -, and gamma -subunit mRNAs of epithelial Na+ channel along rat respiratory tract. Am. J. Physiol. 272 (Cell Physiol. 41): C131-C141, 1997[Abstract/Free Full Text].

17.   Finkel, Y., A. Eklof, and A. Aperia. Mechanisms for colonic sodium transport during ontogeny: loss of an amiloride-sensitive sodium pathway. Pediatr. Res. 24: 46-49, 1988[Abstract].

18.   Fromm, M., F. D. Schulzke, and U. Hegel. Control of electrogenic Na+ absorption in rat late distal colon by nanomolar aldosterone added in vitro. Am. J. Physiol. 264 (Endocrinol. Metab. 27): E68-E73, 1993[Abstract/Free Full Text].

19.   Henning, S. J. Plasma concentrations of total and free corticosterone during development in the rat. Am. J. Physiol. 235 (Endocrinol. Metab. Gastrointest. Physiol. 4): E451-E456, 1978[Abstract/Free Full Text].

20.   Hummler, E., P. Barker, J. Gatzy, F. Beermann, C. Verdumo, A. Schmidt, R. Boucher, and B. C. Rossier. Early death due to defective neonatal lung liquid clearance in alpha ENaC-deficient mice. Nat. Genet. 12: 325-328, 1996[Medline].

21.   Jenkins, H. R., T. R. Fenton, N. McIntosh, M. J. Dillon, and P. J. Milla. Development of colonic sodium transport in early childhood and its regulation by aldosterone. Gut 31: 194-197, 1990[Abstract].

22.   Knowles, M., G. Murray, J. Shallal, F. Askin, V. Ranga, J. Gatzy, and R. Boucher. Bioelectric properties and ion flow across excised human bronchi. J. Appl. Physiol. 56: 868-877, 1984[Abstract/Free Full Text].

23.   Legris, G. J., P. C. Will, and U. Hopfer. Inhibition of amiloride-sensitive sodium conductance by indoleamines. Proc. Natl. Acad. Sci. USA 79: 2046-2050, 1982[Abstract].

24.   Lingueglia, E., S. Renard, R. Waldmann, N. Voilley, G. Champigny, H. Plass, M. Lazdunski, and P. Barbry. Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone. J. Biol. Chem. 269: 13736-13739, 1994[Abstract/Free Full Text].

25.   Martin, C. E., M. H. Cake, P. E. Hartmann, and I. F. Cook. Relationship between foetal corticosteroids, maternal progesterone and parturition in the rat. Acta Endocrinol. 84: 167-176, 1977[Medline].

26.   Mason, R. J., M. C. Williams, J. H. Widdicombe, M. J. Sanders, D. S. Misfeldt, and L. C. Berry, Jr. Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proc. Natl. Acad. Sci. USA 79: 6033-6037, 1982[Abstract].

27.   Matsushita, K., P. B. McCray, Jr., R. D. Sigmund, M. J. Welsh, and J. B. Stokes. Localization of the epithelial sodium channel (rENaC) subunit mRNAs in adult rat lung by in situ hybridization. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L332-L339, 1996[Abstract/Free Full Text].

28.   McCray, P. B., Jr., J. D. Bettencourt, J. Bastacky, G. M. Denning, and M. J. Welsh. Expression of CFTR and a cAMP-stimulated chloride secretory current in cultured human fetal alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 9: 578-585, 1993[Medline].

29.   McCray, P. B., Jr., S. Goodno, R. W. Graeff, F. J. McDonald, M. P. Price, and M. J. Welsh. Ontogeny and regulation of expression of the amiloride-sensitive epithelial sodium channel (hENaC) subunits in human lung (Abstract). Pediatr. Pulmonol. 12: 196-197, 1995.

30.   McDonald, F. J., P. M. Snyder, P. B. McCray, Jr., and M. J. Welsh. Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L728-L734, 1994[Abstract/Free Full Text].

31.   Monder, C., and P. C. White. 11beta -Hydroxysteroid dehydrogenase. Vitam. Horm. 47: 187-271, 1993[Medline].

32.   Murphy, B. E. P. Ontogeny of cortisol-cortisone interconversion in human tissues: a role for cortisone in human fetal development. J. Steroid Biochem. 14: 811-817, 1981[Medline].

33.   O'Brodovich, H., C. Canessa, J. Ueda, B. Rafii, B. C. Rossier, and J. Edelson. Expression of the epithelial Na+ channel in the developing rat lung. Am. J. Physiol. 265 (Cell Physiol. 34): C491-C496, 1993[Abstract/Free Full Text].

34.   O'Brodovich, H., V. Hannam, M. Seear, and J. B. M. Mullen. Amiloride impairs lung water clearance in newborn guinea pigs. J. Appl. Physiol. 68: 1758-1762, 1990[Abstract/Free Full Text].

35.   O'Brodovich, H., B. Rafii, and M. Post. Bioelectric properties of fetal alveolar epithelial monolayers. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L201-L206, 1990[Abstract/Free Full Text].

36.   Olver, R. E., C. A. Ramsden, L. B. Strang, and D. V. Walters. The role of amiloride-blockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J. Physiol. (Lond.) 376: 321-340, 1986[Abstract].

37.   Pacha, J., I. Pohlova, and P. Karen. Regulation of amiloride-sensitive Na+ transport in immature rat distal colon by aldosterone. Pediatr. Res. 38: 356-360, 1995[Abstract].

38.   Pacha, J., M. Popp, and K. Capek. Amiloride-sensitive sodium transport of the rat distal colon during early postnatal development. Eur. J. Physiol. 409: 194-199, 1987[Medline].

39.   Rao, A. K., and G. R. Cott. Ontogeny of ion transport across fetal pulmonary epithelial cells in monolayer culture. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L178-L187, 1991[Abstract/Free Full Text].

40.   Renard, S., N. Voilley, F. Bassilana, M. Lazdunski, and P. Barbry. Localization and regulation by steroids of the alpha , beta , and gamma  subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney. Eur. J. Physiol. 430: 299-307, 1995[Medline].

41.   Sapolsky, R. M., and M. J. Meaney. Maturation of the adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period. Brain Res. Rev. 11: 65-76, 1986.

42.   Stewart, P. M., B. A. Murry, and J. I. Mason. Type 2 11 beta -hydroxysteroid dehydrogenase in human fetal tissues. J. Clin. Endocrinol. Metab. 78: 1529-1532, 1994[Abstract].

43.   Stokes, J. B., and R. D. Sigmund. Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity. Am. J. Physiol. 274 (Cell Physiol. 43): C1699-C1707, 1998[Abstract/Free Full Text].

44.   Strang, L. B. Fetal lung liquid: secretion and reabsorption. Physiol. Rev. 71: 991-1016, 1991[Free Full Text].

45.   Tchepichev, S., J. Ueda, C. Canessa, B. C. Rossier, and H. O'Brodovich. Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am. J. Physiol. 269 (Cell Physiol. 38): C805-C812, 1995[Abstract].

46.   Venkatesh, V. C., and H. D. Katzberg. Glucocorticoid regulation of epithelial sodium channel genes in human fetal lung. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L227-L233, 1997[Abstract/Free Full Text].

47.   Voilley, N., E. Lingueglia, G. Champign, M.-G. Mattei, R. Waldmann, M. Lazdunski, and P. Barbry. The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc. Natl. Acad. Sci. USA 91: 247-251, 1994[Abstract].

48.   Volk, K., R. D. Sigmund, P. M. Snyder, F. J. McDonald, M. J. Welsh, and J. B. Stokes. rENaC is the predominant Na+ channel in the apical membrane of rat renal inner medullary collecting duct. J. Clin. Invest. 96: 2748-2757, 1995[Medline].

49.   Wang, Z., M. Yasui, and G. Celsi. Differential effects of glucocorticoids and mineralocorticoids on the mRNA expression of colon ion transporters in infant rats. Pediatr. Res. 38: 164-168, 1995[Abstract].

50.   Wilcox, J. N. Fundamental principles of in situ hybridization. J. Histochem. Cytochem. 41: 1725-1733, 1993[Abstract/Free Full Text].


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