INTRODUCTION

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There is a growing body of evidence that an appropriate amount of Na⁺ transport-driven removal of liquid by the respiratory epithelium is essential for lung homeostasis (reviewed in 1). Abnormal Na⁺ transport is implicated in the pathogenesis of cystic fibrosis where the lack of interaction of CFTR and ENaC protein leads to increased Na⁺ transport resulting in viscous airway lining fluid (2). In contrast, defective Na⁺ transport may prevent normal postnatal adaptation of the lung of the term infant and worsen respiratory distress of the premature infant (1), or impede edema fluid clearance in congestive heart failure and adult respiratory distress syndrome (3).

Under normal conditions the rate-limiting step in Na⁺ transport is the activity of apical membrane Na⁺ permeant ion channels. One of the Na⁺ channels, epithelial Na channel (ENaC), is amiloride-sensitive and consists of three subunits whose exact stoichiometry in biologic membranes is currently under investigation (4). When expressed together, all three subunits are likely involved in pore formation (8); however, dimers can form in some expression systems and yield channels with differing kinetics (9, 10).

To improve our understanding of human ENaC (hENaC) expression in human respiratory epithelium we quantitated , , and  subunit mRNA of the hENaC in nasal, bronchial, and most distal lung epithelium. It is important to make these measurements in humans since it is known that the relative amounts of ENaC mRNA vary between species (11, 12). A recently developed method based on competitive quantitative reverse-transcriptase-polymerase chain reaction (QRT-PCR) was used for the measurement of mRNA of the samples (13). Additional experiments applying in situ hybridization were conducted to define the cellular distribution of the -hENaC mRNA in the lower airways and distal lung epithelium.

	METHODS

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Subjects

The study group consisted of seven male patients 49 to 66 yr of age who underwent lung lobectomy secondary to cancer at The Toronto Hospital and the Mount Sinai Hospital, Toronto, Canada. Five of the patients were smokers, one had stopped smoking 30 yr earlier, and one was a nonsmoker. Two patients were not receiving medication, and none of the patients was receiving glucocorticosteroid therapy or other medication known to affect ENaC expression (12, 14). The patient data and medications are summarized in Table 1.

The study protocol was approved by the relevant human subject experimental review committees. An informed consent was obtained from the study participants.

Tissue Samples

The nasal samples were obtained under direct vision by scraping the inferior turbinate using a Rhinoprobe (Arlington Scientific, Arlington, TX). Airway epithelium between second- and third-generation bronchi was isolated under direct vision of the excised lobe using the Rhinoprobe. Distal lung was sampled by excising normal tissue adjacent to the pleural surface of the removed lobe. The tissue samples used in the analyses are shown in Table 2.

Molecular Methods

RT-PCR for hENaC subunit mRNA quantitation. Similar to our previous report (13) the tissue specimens were immediately homogenized after collection and stored in RLT lysis buffer (RNeasy RNA preparation kit; Qiagen, Santa Clarita, CA). Total RNA was isolated with the QIAshredder and RNeasy kit according to the manufacturer's instructions, and quantitated by slot blot analysis alongside known amounts of NC1 H661-cell RNA. The total RNA was calculated after hybridization with [³²P]labeled cDNA probe complementary to 18S rRNA.

A truncated fragment of -, -, -hENaC or CK18 cRNA was used as a competitive internal standard during QRT-PCR. The generation of the deletion constructs and the quantitation procedure have been described in detail previously (13). The ENaC expression of individual samples was normalized by dividing it by the CK18 mRNA content of each sample (hENaC: CK18, attomole [amol] per femtomole [fmol]).

The -hENaC cRNA probe for in situ hybridization was generated using as template a 319-bp cDNA fragment (nt, 2169 to 2488) of the 3'UTR region of the human -hENaC subcloned into pGEM3Zf (^+/) vector. The in vitro transcription of this template was performed in the presence of digoxigenin-labeled UTP to allow detection of the mRNA in the tissue samples (DIG Nucleic Acid Detection System; Boehringer Mannheim, Dorval, PQ, Canada).

In situ hybridization studies for localization of -hENaC mRNA were performed on lung tissue that had been fixed in 10% formalin and embedded in paraffin using routine methods. Serial sections of the lungs were mounted on glass, heated under pressure in 0.1 M TRIS-HCl, at pH 8.0, and subjected to in situ hybridization (15). The procedure was carried out using the digoxigenin-labeled sense or antisense cRNA probe to -hENaC. Detection was performed after incubation with alkaline-phosphatase-conjugated anti-DIG antibody, using nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the substrate.

Tissue blocks undergoing in situ hybridization were evaluated for the presence of the epithelial cell marker cytokeratin 18 (CK18). CK protein was detected using the mouse monoclonal antibody CAM5.2 against low molecular weight CK (Becton Dickinson Immunocytometry Systems, San Jose, CA). The anti-CK antibody visualization was enhanced by standard techniques using biotinylated horse antimouse IgG (Vector Laboratories, Burlingame, CA) at 5 L/ml and Elite Avidin Biotin Complex (Vector Laboratories). The reaction was visualized using a diaminobenzidine substrate.

In the last set of experiments the validity of the quantitative RT-PCR method was tested by comparing the data from QRT-PCR against the information obtained from Northern blotting. The Northern blots were produced by standard techniques from total peripheral lung RNA of six subjects. Because of exhaustion of the total RNA stock by the initial determinations described above, only three of these samples were derived from subjects included in our original QRT-PCR data set. The Northern blot was cohybridized with [³²P] labeled cDNA probes to - and -hENaC, corresponding to the regions amplified in the QRT-PCR assay. The probes were of equal specific activity. The ratio of -hENaC/-hENaC across this set of six samples was 1.92 (data not shown), which was deemed comparable to the ratio of 1.75 obtained using QRT-PCR (described below). Unfortunately, Northern blots hybridized with the corresonding -hENaC cDNA probe gave a high background signal, particularly to ribosomal RNA. This obstacle precluded its use as a hybridization probe, but not in PCR since the flanking primers amplify only -hENaC, not rRNA.

Statistical Analysis

The QRT-PCR assays were done in duplicate or triplicate; the average was used as a single datum for the final calculations. The results are expressed as mean ± SD. Analysis of variance with Fisher's post-hoc test was used to determine statistical difference, and p < 0.05 was considered significant.

	RESULTS

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We detected -, -, -hENaC and CK18 mRNA in the human nasal, bronchial, and peripheral lung epithelium. Results are presented in Figure 1. In the nasal epithelium the amount of -hENaC was 18.3 ± 4.7 amol/fmol CK18 mRNA, and this exceeded the amount found in either the bronchial (7.9 ± 1.7 amol/fmol, p < 0.05) or the distal (9.2 ± 1.7 amol/fmol, p < 0.05) lung specimens. In the nasal epithelium, -hENaC mRNA predominated over -hENaC (6.1 ± 2.9 amol/fmol p < 0.05) and -hENaC (0.9 ± 0.2 amol/fmol, p < 0.05).

View larger version (14K): [in this window] [in a new window]   Figure 1.   Quantitative data on -, -, and -hENaC mRNA recovered in airway biopsy specimens that were obtained from seven male patients undergoing lobectomy. The amount of each of the hENaC subunit mRNAs in the nasal, bronchial, and peripheral lung tissue was normalized against the content of CK18. The bars represent the means ± SD. *Difference of nasal -hENaC mRNA from that of the bronchial and peripheral lung (p < 0.05); difference of -hENaC from - and -hENaC in the nasal epithelium (p < 0.05); #difference of nasal -hENaC from that of the bronchial and peripheral lung (p < 0.05).

There were no statistically significant differences in the levels of -hENaC mRNA in the different regions of respiratory epithelium that were studied. In contrast, the expression of -hENaC mRNA increased to 4.6 ± 1.0 amol/fmol in the bronchi and to 4.8 ± 1.9 amol/fmol in the distal lung of the patients (both p < 0.05 against the nasal value). The amount of CK18 mRNA per microgram of total RNA was similar in the nasal, bronchial, and distal lung (0.92 + 0.41, 0.68 + 0.19, and 0.60 + 0.51 fmol, respectively; p = NS).

Because these differences in hENaC subunit mRNAs may be associated with differences in function, the relative proportions of - and -hENaC mRNAs versus the -subunit mRNA in each region were calculated. In the nasal epithelium the amount of -hENaC was 32 ± 8.7% of the level of -hENaC expression. This ratio increased markedly in the bronchus where -hENaC/ -hENaC ratio was 140 ± 48% (p < 0.05) whereas in the distal lung -hENaC was expressed at a level similar to that of the nasal epithelium. The -hENaC/-hENaC ratio was 4.8 ± 3.4% in the nasal epithelium, but it increased more than tenfold in the bronchus and the distal lung to levels of 58.6 ± 11.3% (p < 0.05) and 56.1 ± 30.1% (p < 0.05), respectively.

The in situ hybridization studies of the lungs (Figure 2) demonstrated that -hENaC mRNA was strongly expressed in the cells of the submucosal glands. Expression was patchy in epithelial cells of large airways but uniform in small airways epithelia; -hENaC was also detected in discrete cells of the alveolar regions. These expression patterns are consistent with our recent observations in full term human infants (16).

View larger version (102K): [in this window] [in a new window]   Figure 2.   Sections representative of large (I, Patient 3) and small (II, Patient 2) airways and alveolar region (III, Patient 2). In situ hybridization to detect -hENaC mRNA with antisense (AS) and sense (S) cRNA probes demonstrated specific staining in submucosal glands (arrow) of large airways, and uniformly in epithelial cells of small airways (a). No staining was found in blood vessel epithelium. Immunocytochemistry for low molecular weight cytokeratins-(CK)-labeled epithelial cells linig all respiratory tract.

	DISCUSSION

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Our findings have demonstrated that in humans the level of ENaC subunit mRNA varies along the respiratory tract. The mRNA content of the hENaC subunits in the human nasal epithelium was similar to that reported in healthy male nonsmokers (13). The present investigation is the first to simultaneously quantitate ENaC mRNA in the upper and lower airways and distal lung regions. We report that the amount of - and -hENaC mRNA relative to the -subunit levels increases from the proximal to distal regions of the respiratory tract. This is in harmony with the findings in the rat model where the - and -ENaC subunit mRNAs increased toward the bronchi and bronchioli (17) and were dependent on whether the subunit was expressed in the airway epithelium, alveolar cells, or the mucosal glands (18).

Previous workers have shown that the amount of amiloride-sensitive potential difference (PD) across the respiratory epithelium decreases from the nasal to the bronchial regions of the respiratory tract (19). We observed a comparable decrement in -hENaC mRNA expression with the abundant -hENaC mRNA content in the nasal epithelium decreasing toward the distal airways and air spaces. We also demonstrate that the - to -hENaC mRNA ratio is tenfold higher in the nasal than in the distal lung epithelium. Accordingly, it may be speculated that there is a negative correlation between the ratio of -hENaC/-hENaC mRNA levels and transepithelial PD. First, a precedent observation in humans supports this assumption. Otulakowski and coworkers (13) demonstrated in a group of healthy adult men that nasal PD increased with decreasing -hENaC mRNA levels. When the data from the previous study (13) are used to calculate the -hENaC/-hENaC ratio, and plotted against the nasal turbinate PD, a significant inverse correlation is established (r² = 0.7, p < 0.05, regression analysis, not shown). Second, recent data have demonstrated that high aldosterone levels known to increase epithelial amiloride-sensitive Na⁺ channel activity and mRNA in vitro (14) were associated with an increased renal -ENaC protein in the rat and reduction in abundance and size of the -ENaC protein (22). Thus, the data on transepithelial PD and the present findings on ENaC subunit mRNA ratios along the human respiratory tract epithelium are in harmony with recent observations of increased channel function in the presence of increased -ENaC and decreased -ENaC.

The present observations were limited to measurement of mRNA and do not allow conclusion about the actual subunit stoichiometry of the ENaC channel protein. A direct relationship between mRNA and functional protein cannot be predicted, and it is possible that ENaC function is regulated by post-transcriptional mechanisms. The existence of alternative 5'untranslated regions (UTRs) in mRNA for hENaC (23) suggests the potential for translational regulation of ENaC through regulation of expression of the alternative UTRs. The rapid turnover of the -hENaC and -ENaC protein in an epithelial membrane (24) may be another way to regulate the channel activity. Other alternate potential sites of regulation include: ENaC subunit assembly and stability, ENaC transport to the membrane, and/or control of ENaC function at the membrane surface. Thus an assessment of protein expression, if suitable antibodies become available in the future, will aid in the elucidation of the role of translational regulation.

We and others have consistently found ENaC in loci where functional Na⁺ transport takes place. However, parallel measurements of the ENaC subunit proteins and amiloride sensitive PD will be necessary to unravel the relative expression of different ENaC subunits in vivo to transepithelial Na⁺ transport. Our finding of the profound and independent variation of ENaC subunit gene expression along with the known variation in amiloride-sensitive PD, may imply that the channel composition and PD may differ in a consistent way.

We have now demonstrated in male patients in steady clinical state that the proportion -hENaC mRNA relative to the - and -subunits changes along the range of the respiratory epithelium. In future studies it will be interesting to find out whether the subunit composition of lung ENaC responds to acute conditions where fluid accumulates on the luminal side of the epithelium.