Main

The amiloride-sensitive sodium channel is a channel expressed in epithelial cells of sodium-reabsorbing tissues such as the kidney collecting duct, the distal colon, the duct of several exocrine glands, the lungs, and airway(1, 2). These channels, regulated by hormones such as aldosterone and vasopressin(1, 2), play a major role in the control of sodium homeostasis and blood pressure. Recently, functional constituents of the amiloride-sensitive sodium channel have been cloned from a rat distal colon cDNA library and named α, β, andγ rENaC for: rat epithelial Na c hannel(3, 4). The αENaC subunit expressed alone in Xenopus laevis oocytes, but not the β or theγ subunit, can drive sodium absorption, suggesting that αENaC is crucial for functioning of the channel and could lie in its pore-forming region(3). Furthermore, mutation studies have shown that the first and especially the second transmembrane domains of αENaC are very important for conference of their electrophysiologic and pharmacologic properties to the channel(5). The β and γ subunits have been demonstrated to stabilize the channel and allow proper insertion into the cytoplasmic membrane(4, 6). Expression of the three subunits in X. laevis oocytes results in a 100-fold increase in the amiloride-sensitive sodium current compared with theαrENaC subunit alone(4). The β and γ subunits also regulate the activity of the channel inasmuch as their mutations have been associated with Liddle's syndrome, a rare familial form of hypertension(7, 8).

The channel could have a significant role to play in the control of thickness of secretions in the airways(9) and in regulating the amount of liquid lining the alveoli(10, 11), because the process is highly dependent on Na+ transport. Work by our group and others has shown that sodium transport is involved in the reabsorption of pulmonary edema(12–14). Sodium transport is also involved, late in gestation and around birth, in the clearance of liquid secreted in alveoli during fetal life(15, 16). The early death of αmENaC-deficient mice, which are unable to clear liquid from their lungs, points to the importance of αENaC in the lungs around birth(17). Modulation of expression of theα subunit of the sodium channel has been detected in the rat lung late during gestation and at birth(18, 19). We do not know, however, if the regulation of αENaC expression around birth is the same in all tissues that play a significant role in Na+ transport and homeostasis.

In the present study, we have cloned part of the mouse epithelial sodium channel cDNA and investigated the modulation of αmENaC expression in the lung, distal colon, and kidney during mouse development and around the time of birth. We found that, for a given tissue, the expression profile ofαmENaC mRNAs is very similar to the expression of α1 Na+-K+-ATPase mRNAs, a constituent of the sodium pump involved in transepithelial sodium transport. The expression of αmENaC is modulated differently in the lungs and the kidneys during development and at birth. There is up-regulation of αmENaC and α1 Na+-K+-ATPase mRNA expression at birth which is not detected in the kidney. There is also developmental regulation of the size of the transcript in the distal colon where a short 1.2 kb mRNA, deleted of the 5′ part of the transcript, is replaced by the mature 3.5-kb transcript after birth.

METHODS

Experimental Protocols

Cloning of αmENaC cDNA. The α, β and γ subunits of the amiloride-sensitive sodium channel are part of a new family of ionic channels that encompasses Caenorhabditis elegans degenerins(20, 21). For the purpose of this study, we chose to amplify, by reverse transcription-PCR, a DNA probe coding for αmENaC that we could use for Northern blot hybridization. Degenerate primers were designed for amplification equivalent to the degenerin/αrENaC type of channel. We found that two regions, the first in the cysteine-rich domain, which codes for the HSCFQE sequence in αrENaC (His-445 to Glu-450), and the FFKELN sequence (Phe-554 to Asn-559), 10 amino acids outside the second hydrophobic domain (M2), were highly conserved between mec-4(22), deg-1(23), and αrENaC(3) (Fig. 1). A degenerate oligonucleotide at the position of the HSCFQE sequence of αrENaC was designed for the sense primer: 5′-GGGGATCCCI(C/T)TC(C/T)TGCTTCCA(A/G)IA-3′(Fig. 1). A BamHI site was also included at the 5′ end of the oligonucleotide to facilitate subsequent cloning of the PCR-amplified fragment. For the antisense primer, we used a sequence at the position of the FFKELN sequence of αrENaC, flanked by a SstI site: 5′-CCGAGCTCTTCAGIT(G/C)CT(T/C)G(A/T)AGAAIA-3′(Fig. 1). A 344-bp fragment was successfully amplified(αmENaC nt 1333-1676) and cloned, employing cDNA from mouse kidneys as template.

Figure 1
figure 1

(A) Schematic representation of the α subunit of the amiloride-sensitive sodium channel. The two hydrophobic domains(M1, M2) and the cysteine-rich region (hatched box) are depicted. The arrowheads show the position of the degenerate oligonucleotides used for PCR amplification of the sodium channel.(B) Comparison of DNA sequences between the homologous regions ofαrENaC and the degenerins deg-1 and mec-4 of the nematode C. elegans. The degenerate primers used for PCR amplification and cloning ofαmENaC are also shown.

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Regulation of αmENaC expression during mouse development. For this study, the lungs, kidneys, and distal colon of CD1 mice were collected at different periods late during gestation (d 15.5, 16.5, 17.5, 18.5, and 19), at birth (2 h), after birth (d 1, 3, and 5), and in adulthood. The stage of development of CD1 mouse embryos was deduced by the time of gestation (±12 h) and by morphometric and morphologic criteria(24, 25). Total RNAs from the three tissues were extracted, electrophoresed on 1% agarose formaldehyde gels, and subjected to Northern blotting. The blots were hybridized for 16 h at 62 °C with the 344-bp αmENaC cDNA fragment labeled by random priming with[α-32P]dCTP. During gestation, the lungs, kidneys, and distal colons of several embryos were pooled to give sufficient material (40 mg) for RNA extraction. For the time points studied after birth, each lane of the gel came from a distinct animal except for that of the distal colon, in which the tissues of several mice were pooled.

To determine whether other components of the Na+ transport system were regulated similarly to αmENaC around the time of birth, the Northern blots were also hybridized with probes for the α1 subunit of Na+-K+-ATPase(26), a gift from Dr. J. Orlowski (Physiology Department, McGill-University, Montreal, Canada), which consists of a NarI-StuI 332-bp fragment coding from nt 89 to 421 (from the 5′-untranslated domain to Arg-61) of the rat kidney and brain α isoform(27).

To perform a quantitative study of the expression of αmENaC andα1 Na+-K+-ATPase mRNAs in the lungs and kidneys around the time of birth, three RNA samples from different animals or different pools of embryonic lungs or kidneys were studied from d 16.5 of development to 5-d-old pups and adults and subjected to densitometric quantification with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).αmENaC expression in each lane of Northern blots was normalized with a 18 S rRNA probe to ensure that the same amount of RNA was loaded onto each well. The rat 18 S rRNA probe was generated by PCR and purified by gel electrophoresis. It consists of a 640-bp fragment amplified between nt 852 and 1492 of the rat 18 S rRNA sequence(28).

Determination of the sequence conserved by the αmENaC 1.2-kb transcript detected in the distal colon. To map the short (1.2 kb)αmENaC transcript detected in the mouse distal colon, Northern blots were hybridized sequentially with probes recognizing distinct segments ofαmENaC mRNA. Because of great homology between the mouse and rat sequence, other αmENaC cDNAs were amplified by reverse transcription-PCR and cloned in the pCR™II vector (Invitrogen, San Diego, CA) usingαrENaC sequence as primers. A 1.6-kb 5′ αmENaC cDNA(αmENaC nt 76-1676) from nt 76 to 1676 (Met-26 to Asn-559) was amplified by PCR with the 5′-ATG AAG GGC AAC CAA TTC AAG GAG-3′ sense primer and the antisense primer used to clone the 344-bp αmENaC probe(αmENaC nt 1333-1676). The 5′ clone (αmENaC nt 76-1676) was further digested with KpnI to produce two probes of 757 bp(αmENaC nt 76-833) and 843 bp (αmENaC nt 833-1676). The 3′-coding region of αmENaC (αmENaC nt 1333-2097) from nt 1333 to 2097 (His-445 to stop codon) was amplified by PCR with the sense primer used to clone the 344 αmENaC probe (αmENaC nt 1333-1676) and the following antisense primer: 5′-TCA GAG CGC CGC CAG GGC ACA GGC-3′. These clones were partially sequenced to confirm the identity of the cDNA.

We also used αrENaC cDNA generously given to us by Dr. B. C. Rossier(Institut de Pharmacologie et de Toxicologie, Université de Lausanne, Lausanne, Switzerland)(3, 4) to complete mapping of the 1.2-kb αmENaC transcript. The 5′-coding and noncoding region of αmENaC was detected with 303-bp EcoRI digestion ofαrENaC cDNA (αrENaC 0-223). The 3′-untranslated region of mRNA was detected with two probes derived by digestion of αrENaC cDNA subcloned in pcDNA 3 vector (Invitrogen). ApaI digestion leaves a probe (αrENaC nt 2232-3004) recognizing most of the 3′-untranslated region of mRNA between nts 2232 and 3004. The Hin DIII-XbaI fragment (αrENaC nt 2702-3004) recognizes a 302-bp fragment localized in the 3′ more distal part ofαmENaC mRNA from nt 2702 to 3004.

Methodology

PCR amplification of αmENaC. For cDNA synthesis, total RNA [7 μg (9 μL)] was incubated at 65 °C for 5 min with 1 μL of oligo(dT)12-18 (100 μg/mL). After 3 min of incubation on ice, the RNAs were incubated for 60 min at 37 °C in reverse transcription buffer(50 mM Tris-HCL, pH 8.3, 75 mM KCl, 3 mM MgCl2) (Life Technologies, Inc., Gaithersburg, MD) and a mixture of dNTPs (0.5 mM each), human placental RNase inhibitor (0.5 μL) (5 U), and 1 μL (200 U) of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The reverse transcriptase was subsequently inactivated at 95 °C for 10 min, and the cDNA used for PCR was frozen at -40 °C.

For PCR amplification, 8 μM sense oligonucleotide (1 μM (50 pmol) for each possible oligonucleotide) and 8 μM antisense oligonucleotide were added to the PCR buffer (20 mM Tris-HCl, pH 8.5, 1.5 mM MgCl2, 50 mM KCl, 0.1% (vol/vol) Tween 20) along with dNTPs (0.2 mM) and 0.2 μL (1 U) of Taq polymerase (Life Technologies, Inc.). The PCR conditions were as follows: 40 cycles of 1 min 94 °C denaturation, 2 min 50 °C annealing, and 3 min 72 °C extension.

Cloning and sequencing of αmENaC cDNA. PCR products were separated on 1% agarose gel in 0.5 × TBE, and the bands were excised and purified with “Gene Clean” (Bio 101, Vista, CA). For cloning the PCR fragment, pBluescript KS+ vector (Stratagene, La Jolla, CA) and PCR DNA were double digested with BamHI and SstI, ligated overnight, and transformed in JM105 cells. The clones were sequenced by the dideoxy technique(29) using T7 DNA polymerase (United States Biochemical Corp., Columbus, OH).

RNA extraction and Northern blotting. The Northern blot used to detect αmENaC transcripts in adult mouse tissues was purchased from Clontech (Palo Alto, CA) and contained 2 μg of poly(A+) mRNA per lane. For the developmental study, RNA from CD1 mouse tissues was purified by a modification of the guanidinium-phenol method(30). The lungs, kidneys, and distal colon of embryos and young and adult mice were harvested, and 40 mg of tissues were minced with scissors in 600 μL of ice-cold solution D(30). The samples were then homogenized in Eppendorf tubes with the sterile piston of a 1-mL syringe and processed as described elsewhere(30). Ten micrograms of total RNAs were electrophoresed on 1% agarose-formaldehyde gel and transferred to a Hybond N+ nylon membrane (Amersham Corp., Arlington Heights, IL) by overnight blotting with 10 × SSC. After fixing RNAs to the membrane by baking the blots at 80 °C for 2 h, the membranes were prehybridized for 2 h and hybridized for 16 h at 62 °C in Church buffer [0.5 M sodium phosphate, pH 7.2, 7% SDS (wt/vol), 1 mM EDTA, pH 8](31) with the αmENaC probe labeled by random priming with [α-32P]dCTP. After hybridization, the membranes were washed successively for 30 min with 100 mM sodium phosphate (pH 7.2), 0.1%(wt/vol) SDS, 40 mM sodium phosphate (pH 7.2), 0.1% (wt/vol) SDS, and 40 mM sodium phosphate (pH 7.2), 1% (wt/vol) SDS. Blots were exposed to Kodak Xar film, using an intensifying screen, or to the PhosphorImager (Dupont) for densitometric analysis. For stripping of the membranes, the blots were incubated with 0.1 × SSC and 1% SDS at 95 °C, then cooled for 30 min to room temperature. The membranes were rinsed with 5 × SSC and rehybridized.

Statistical analysis. To determine the statistical significance of the expression of αmENaC and α1 Na+-K+-ATPase mRNA detected at birth in the lungs and kidneys, the means of the densitometric values found at birth (2 h) were analyzed by unpaired t test with data at d 19 of gestation and at d 1 (24 h).p < 0.05 was considered to be significant. All values are given as means ± SEM.

RESULTS

PCR Amplification and cloning of a probe for αmENaC. Using degenerate oligonucleotides designed in conserved regions ofαrENaC and C. elegans degenerins mec-4 and deg-1(Fig. 1), we amplified by reverse transcription-PCR and cloned a 344-bp fragment of αmENaC (αmENaC nt 1333-1676). Mouse kidney cDNA served as template. The sequence of the αmENaC amplified fragment showed strong homology with the rat (αrENaC) and humans(αhENaC) (Fig. 2). Homology with the rat sequence was 92% for DNA and 95% for amino acids. It dropped to 83% for DNA and 78% for amino acids with the human sequence.

Figure 2
figure 2

Comparison of the 344-bp αmENaC clone (mouse) with the corresponding DNA sequence of αrENaC (nt 1333-1676) (rat)(3) and αhENaC (nt 1252-1595) (human)(19). The translated amino acid sequence of αmENaC is also depicted. The conserved nts with the mouse sequence are depicted by a dash (-). There is 92% identity of the DNA sequence between mice and rats and 83% identity between mice and humans. The GenBank™ accession no. for theαmENaC sequence is U52006.

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αmENaC expression in the mouse. The 344-bp αmENaC clone was used as a probe to hybridize a Northern blot consisting of 2μg/lane of poly(A+) RNA from various adult mouse tissues. A 3.5-kb transcript was highly expressed in the kidneys and lungs (Fig. 3). A signal was also detected in the liver (Fig. 3). A Northern blot using 10 μg of total RNA per lane allowed the detection of a 3.5-kb αmENaC transcript in the mouse distal colon as well (Fig. 4B).

Figure 3
figure 3

αmENaC expression in various mouse tissues. The membrane containing 2 αg of poly(A+) RNA per lane was hybridized with the 344-bp αmENaC probe. A 3.5-kb transcript is detected in the kidney and lung. Smaller RNA bands are also detected in the liver.

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Figure 4
figure 4

αmENaC expression in the lung, kidney, and distal colon during mouse development. Ten micrograms of total RNA were loaded onto each lane, the gels were subjected to Northern blotting, and the membranes were hybridized successively with αmENaC, α1 Na+-K+-ATPase, and 18 S rRNA probes. In the three tissues,αmENaC expression starts on d 17.5 of gestation. (A) Lungs and kidneys were collected on d 16.5 (E16.5), 17.5 (E17.5), 18.5 (E18.5), and 19 (E19) of gestation, 2 h after birth(NB), after 1 d (1), 3 d (3), 5 d (5) of postnatal life and in adulthood (AD). The expression of αmENaC and α1 Na+-K+-ATPase mRNAs during development seems to be regulated similarly in the lungs and kidneys. A transient increase in the expression of αmENaC mRNAs and a slight increase in α1 Na+-K+-ATPase mRNAs are detected at birth in the lungs. The sizes of the mRNA bands detected are, respectively, 3.5 kb for αmENaC, 3.7 kb for α1 Na+-K+-ATPase, and 1.8 kb for rRNA.(B) αmENaC expression in the distal colon during mouse development. Distal colons were collected on d 15.5 (E15.5) and 17.5(E17.5) of gestation, after 1 d (1), 2 d (2), 5 d (5), 10 d of postnatal life (10), and in adulthood(AD). Beside the 3.5-kb transcript detected in the lung, kidney, and mature distal colon, a 1.2-kb transcript is also seen in the distal colon during gestation and early postnatal life.

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Developmental expression of the sodium channel in the mouse.αmENaC expression starts late during mouse development, at d 17.5 of gestation for the three tissues investigated: the lungs, kidneys, and distal colon (Fig. 4). The expression profile of αmENaC mRNA around the time of birth was different, however, for the three tissues (Fig. 4). Only the lungs showed strong expression ofαmENaC mRNA at the end of gestation and a large transient elevation ofαmENaC mRNA at birth (Fig. 4A). α1 Na+-K+-ATPase mRNA, a subunit of the sodium pump involved in transepithelial sodium transport, showed an expression profile modulated similarly to αmENaC mRNA in the lungs and kidneys. There was a slight increase in the expression of α1 Na+-K+-ATPase mRNA in the lungs at birth that was not detected in the kidneys (Fig. 4A).

Regulation of αmENaC expression in the lungs and kidneys at birth. αmENaC transcripts are regulated differently in the lungs and kidneys at birth. Densitometric quantification of Northern blots showed a 2.5-fold rise in αmENaC mRNA expression in the lungs at birth(NB) compared with adjacent time points: 19 d embryonic(E19), and 1 d postnatal (1) (Fig. 5A). This rise was statistically significant (p < 0.005). There was also a significant 1.5-fold increase (p < 0.05) in expression of theα1 subunit of Na+-K+-ATPase in the lungs at birth, compared with adjacent time points: 19 d embryonic (E19), and 1 d postnatal (1) (Fig. 5B). In contrast, in the kidneys, the expression of αmENaC and α1 Na+-K+-ATPase mRNAs showed no significant increases at birth (Fig. 5, C and D). The expression of αmENaC andα1 Na+-K+-ATPase mRNAs followed a similar profile with a steady increase late during gestation and a plateau after birth.

Figure 5
figure 5

Densitometric quantification of αmENaC andα1 Na+-K+-ATPase mRNA expression in the lungs(A and B) and kidneys (C and D) around the time of birth. The amount of mRNA detected for each lane of the Northern blots was normalized with an 18 S rRNA probe and expressed as arbitrary units of αmENaC (A and C) or α1 Na+-K+-ATPase (B and D) mRNAs. The data are the means ± SEM of a minimum of three independent RNA samples. Embryonic lungs and kidneys were harvested on d 16.5 (E16.5), 17.5(E17.5), 18.5 (E18.5), and 19 (E19) of gestation. Lungs and kidneys from newborn mice (NB) were taken 2 h after birth and, for the other time points, collected after 1 d (1), 3 d (3), 5 d (5), and in adulthood (AD). The asterisk (*) indicates a statistically significant difference(p < 0.005) in lungs between αmENaC expression at birth(NB) and the adjacent time points: d 19 of gestation (E19) and d 1 (24 h) after birth (1) (A). The Northern blots studied in A and C were stripped and rehybridized for expression of the α1 subunit of Na+-K+-ATPase in the lungs (B) and kidneys (D). The asterisk (*) depicts a statistically significant difference (p < 0.05) in lungs between α1 Na+-K+-ATPase expression at birth(NB) and at d 19 of gestation (E19) as well (p< 0.005) as between birth (NB) and d 1 (24 h) postnatal(1) (B). There is also a statistically significant difference (p < 0.05) between d 18.5 of gestation(E18.5) and d 1 postnatal (1) in the kidneys(D).

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Detection of a short 1.2-kb transcript during gestation in the distal colon. In the distal colon, two transcripts of 3.5 and 1.2 kb were detected (Fig. 4B). The short transcript (1.2 kb) was the predominant mRNA detected during gestation, whereas after birth it was replaced by the 3.5-kb mRNA (Fig. 4B). The 1.2-kb transcript could still be detected until d 5 in the distal colon, but on d 10, as in the adult mouse, it was no longer apparent.

To determine which part of αmENaC mRNA was conserved in the short transcript, Northern blots from the distal colon were hybridized sequentially with probes recognizing different segments of αmENaC mRNA (Fig. 6). All the probes, except αrENaC nt 0-223 and αmENaC nt 76-833 at the 5′ portion of mRNA, detected the short transcript. This suggests that the 1.2-kb mRNA lacks the 5′ portion of mRNA coding for the N-terminal cytoplasmic domain, the first transmembrane domain, and part of the extracytoplasmic domain.

Figure 6
figure 6

Characterization of αmENaC sequences conserved in the 1.2-kb transcript detected in the mouse distal colon. The map shows a schematic view of αENaC mRNA and the nature of the cDNA probes used for Northern blot hybridizations. The number in the name of the αENaC probes refers to the position of the first and last nt according to the αrENaC sequence(4). The sequences between the start(AUG) and stop (UGA) codon are in the coding (translated) portion of the mRNA. The hydrophobic domain coding regions (M1, M2) are also depicted (black box). The plus (+) and minus (-) signs of the table signify a positive or negative signal, respectively, after Northern blot hybridization.

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DISCUSSION

In the present study, we have cloned a segment of the α subunit of the mouse epithelial sodium channel, the amiloride-sensitive channel involved in sodium transport by epithelial cells(1, 2). The sequence of αmENaC cDNA shows high homology with rat (92% identity) and human (83% identity) αENaC. Using αmENaC cDNA as a probe, we have studied the regulation of expression of the epithelial channel in the mouse during development and at birth. In the adult mouse, αmENaC transcripts are expressed mainly in the lungs, kidneys, and distal colon. The tissue expression that we find in the mouse is very similar to what has been reported in rats(3, 19), humans(19, 32), and cattle(33). This localization is not surprising, because the kidneys and distal colon are involved in sodium and water reabsorption(1, 2). Sodium transport is also known to occur in the lungs(10, 11), where amiloride-sensitive sodium channels have been demonstrated in primary culture of adult alveolar type II cells(19, 34–36) and in fetal distal lung epithelial cells(37–40). αrENaC has also been detected by in situ hybridization in various locations of the rat lungs: alveolar type II cells of alveoli and epithelial cells of the trachea, bronchi, and bronchioles(41).

αmENaC mRNA expression starts late during mouse development at d 17.5 of gestation for the three tissues investigated: the distal colon, kidneys, and lungs. αmENaC transcripts are expressed continuously thereafter through adulthood in these tissues (Fig. 4). In the mouse lungs, the start of αmENaC expression corresponds to the beginning of the saccular stage of lung development (terminal sac stage)(24, 25), as in the rat(18, 19).

One major difference between the three tissues is the size of the transcripts detected (Fig. 4). In the lungs and kidneys, a 3.5-kb transcript is found from gestation through adulthood. In the mouse distal colon, however, a 1.2-kb transcript is present during gestation and early postnatal life, whereas in later stages the 3.5-kb form is seen. The 3.5-kb transcript in these three tissues corresponds to the size of theαENaC transcript that has been reported in rat lungs and kidneys(3, 19). Variations in the size of αENaC transcripts have already been recorded(19, 32). In humans, besides the major-3.8 kb transcript, a minor 3.4-kb transcript has been detected in the lung and colon(19). McDonald et al.(32). reported the presence of two transcripts, a major 3.9-kb mRNA and a minor 3.2-kb transcript in the human lung, kidney, pancreas, and placenta. Smaller transcripts have also been found in the human liver(19). The 1.2-kb transcript in the mouse distal colon is, however, unique in two ways: its size is very different from that of transcripts detected so far for αENaC. Furthermore, during gestation, the 1.2-kb transcript of αmENaC is not a minor but clearly a major form.

Several mechanisms could allow the generation of two transcripts detected in the mouse distal colon. These transcripts could arise by alternative splicing or the use of different transcriptional start sites or polyadenylation sites. So far, no evidence has been found in the literature to suggest that alternative transcription start sites, or alternative polyadenylation sites, are involved in the modulation of size of αENaC mRNA. However, alternative splicing of αrENaC was detected recently in the rat(42), indicating that a similar event could be involved in the generation of the 1.2-kb transcript in the distal colon. We have mapped the αmENaC transcript detected in the mouse distal colon with probes recognizing all sequences of αENaC mRNA. Our results show that the 1.2-kb transcript is deleted from the 5′ part of the mRNA. Probes recognizing mRNA from the transcription start site to nt 833 do not recognize the 1.2-kb transcript (Fig. 6). This suggests that the short αmENaC transcript lacks the N-terminal cytoplasmic domain, the first transmembrane domain, and part of the extracellular domain. The sodium channel encoded by such a transcript would result most likely in a nonfunctional channel. This was the case for the channel encoded by alternative splicing of αrENaC mRNAs in the rat, which lacks one of the transmembrane domains and was found to be inactive when expressed in Xenopus oocytes(42).

So far, we do not know the physiologic significance of the short (1.2 kb) transcript in the immature distal colon. However, modulation in the size of the αmENaC transcript detected during gestation could be related to the regulation of sodium absorption in the distal colon. The presence of a 1.2-kbαmENaC transcript in the mouse could be a mechanism that prevents amiloride-sensitive Na+ transport in the immature distal colon. It is known that in the distal colon of suckling rats there is induction of amiloride-sensitive Na+ transport(43, 44). This process occurs around 5 d after birth(43), the time when we detect the appearance of the 3.5-kb αmENaC transcript as well as the disappearance of the 1.2-kb transcript. Before birth, the immature distal colon might not need amiloride-sensitive sodium transport and would express the 1.2-kb transcript, leading to a nonfunctional channel. Glucocorticoids, low Na+ uptake, and a high level of aldosterone have been shown to be involved in the regulation of electrogenic Na+ transport detected in the distal colon of suckling rats(43, 44). It is possible then that these factors could also modulate the conversion of 1.2-kb to 3.5-kb αmENaC transcripts in the mouse distal colon.

Although it has been shown that the expression of αENaC mRNAs is regulated differently in the lungs, kidneys, and distal colon of adult rats(45, 46), our work is the first to demonstrate that αmENaC expression is regulated differently in the lungs and kidneys during development. This suggests that different factors regulate the expression of αmENaC mRNAs during development in the lungs and kidneys. Steroids and a diet low or normal in sodium have been shown to controlαrENaC mRNA accumulation in animals and cell culture(18, 45, 47, 48). Among these factors, dexamethasone and thyroid-releasing hormone, given late during gestation, have been found to increase αrENaC expression in the fetal lung(18, 48). Dexamethasone also augmentsαmENaC mRNA expression in cultured fetal lung epithelial cells(47) and in an airway epithelial cell line(49). As dexamethasone does not increase the expression of αrENaC mRNAs in the kidneys(45), it suggests that steroids could be one of the possible factors involved in regulation of the lung-specific accumulation of αmENaC mRNA at birth.

Our results have also allowed us to compare the expression profile ofαmENaC mRNA with that of α1 Na+-K+-ATPase mRNAs during development. They suggest that the expression of αmENaC andα1 Na+-K+-ATPase mRNAs is modulated similarly for a given tissue. However, the pattern of expression of αmENaC andα1 Na+-K+-ATPase mRNAs changes from tissue to tissue. At birth, there is a transient increase (2.5-fold) of αmENaC transcripts in the mouse lungs as well as an augmentation in the expression ofα1 Na+-K+-ATPase mRNAs (1.5-fold). In the kidneys, the expression profiles of αmENaC and α1 Na+-K+-ATPase mRNAs do not show such an increase at birth. Instead, there is a gradual rise in the expression of αmENaC andα1 Na+-K+-ATPase mRNAs during gestation that reaches a plateau after birth. Correlation analysis of the expression ofαmENaC and α1 Na+-K+-ATPase mRNAs confirmed our observations. We found a significant correlation between the expression ofαmENaC and α1 Na+-K+-ATPase mRNAs in the lungs (R2 = 0.767, p < 0.0001). Similarly, there was a significant correlation between the expression of αmENaC andα1 Na+-K+-ATPase mRNAs in the kidneys(R2 = 0.805, p < 0.0001). No correlation was found, however, in the expression of αmENaC mRNAs between the lungs and the kidneys (R2 = 0.06) or in the expression ofα1 Na+-K+-ATPase mRNAs (R2 = 0.092) in the lungs and the kidneys. These results suggest that, for a given tissue, common factors could regulate the expression of αmENaC andα1 Na+-K+-ATPase mRNAs during development.

Our data confirm what has been reported previously for αrENaC expression around birth in the rat lungs(18, 19, 48) and for the expression of the α1 subunit of the sodium pump in rat(50) and mouse(26, 51) lungs. The presence of αENaC has been shown to be essential for clearing liquid from the lungs at birth, because αENaC-deficient mice develop respiratory distress and die shortly after birth(17). The high expression ofαmENaC and α1 Na+-K+-ATPase mRNAs that we detected in the lungs of newborn mice could be part of the mechanisms that modulate sodium transport from lung alveoli at birth. The recent finding of an increase in expression of the AQP-4 water channel during the perinatal period in rat lungs(52) suggests that, at birth, there could be a regulated increase in the expression of several components involved in sodium-driven liquid reabsorption of the lungs.

In conclusion, our work has revealed that, in the mouse, αmENaC expression is complex during development and shows different expression profiles in the lung, kidney, and distal colon. We found coregulation in the expression of αENaC and the α1 subunit of Na+-K+-ATPase mRNAs in the lungs and kidneys and describe developmental regulation of the size of the αmENaC transcript in the distal colon.