Expression of the Na+/H+ and Clminus /HCOminus 3 exchanger isoforms in proximal and distal human airways

P. K. Dudeja, N. Hafez, S. Tyagi, C. A. Gailey, M. Toofanfard, W. A. Alrefai, T. M. Nazir, K. Ramaswamy, and F. J. Al-Bazzaz

Department of Medicine, University of Illinois at Chicago and Westside Veterans Affairs Medical Center, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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Recent studies have indicated the presence of Na+/H+ and Cl-/HCO-3 exchange activities in lung alveolar and tracheal tissues of various species. To date, the identity of the Na+/H+ (NHE) and Cl-/HCO-3 (AE) exchanger isoforms and their regional distribution in human airways are not known. Molecular species of the NHE and AE gene families and their relative abundance in the human airway regions were assessed utilizing RT-PCR and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, and large bronchi and trachea) were harvested for RNA extraction. Gene-specific primers for the human NHE and AE isoforms were utilized for RT-PCR. Our results demonstrated that NHE1, AE2, and brain AE3 isoforms were expressed in all regions of the human airways, whereas NHE2, NHE3, AE1, and cardiac AE3 were not detected. RNase protection studies for NHE1 and AE2, utilizing glyceraldehyde-3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. In contrast, the levels of AE2 mRNA remained unchanged. Differential expression of these isoforms in the human airways may have functional significance related to the airway absorption and secretion of electrolytes.

human lung; anion exchangers; cation exchangers; reverse transcription-polymerase chain reaction; ribonuclease protection


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY EPITHELIA have been suggested to be involved in both fluid and electrolyte absorption and secretion. However, little is known about the mechanisms of fluid and electrolyte secretion and/or absorption in the human airways (8). The distal airways (medium and small terminal bronchioles), which contribute >80% to the total mucus membrane surface area of the conducting airways, are very likely to have a pivotal role in the formation and regulation of airway secretions in the normal lung. Distal airways are also the major sites of lung pathology in chronic bronchitis, asthma, and cystic fibrosis. The characteristics of membrane transport by the distal airway epithelia have not been well defined in mammalian lungs.

Various mechanisms involving translocation of Na+ and Cl- across cell membranes have been identified. These include electrogenic processes, such as Na+ and Cl- channels (40, 42), and electroneutral processes, such as Na+/H+ exchange and Cl-/HCO-3 exchange (2, 39, 45). These mechanisms play a vital role in the regulation of intracellular pH and volume, vectorial transport of these ions, and proton or HCO-3 secretion in various fluids, such as gastric, intestinal, exocrine pancreatic, and renal tubule secretions. Recently, gene families for both the Na+/H+ exchangers (NHE) and Cl-/HCO-3 exchangers or anion exchangers (AEs) have been identified. The roles of individual members of these gene families in basic physiology and pathophysiology of various epithelial and nonepithelial tissues are currently under intense investigation.

The NHE gene family has been shown to include five different isoforms (NHE1 to NHE5), and NHE1, NHE2, and NHE3 isoforms are the most characterized members of this gene family (5, 39, 45). NHE1 is considered to be the ubiquitous isoform localized to the basolateral membranes of the polarized epithelial cells and involved in housekeeping functions, whereas NHE2 and NHE3 isoforms have been considered to be the epithelial isoforms localized to the apical membranes of the polarized epithelial cells. NHE3 has been shown to be an important apical isoform involved in the vectorial Na+ transport in the kidney and intestinal epithelium. The AE gene family includes three structurally and functionally related anion exchangers, AE1, AE2, and AE3 (2). The AE2 isoform has been suggested to be the epithelial isoform, whereas AE1 has been suggested to be erythroid and AE3 has been suggested to be a neuronal homologue (2, 11, 17). Recent studies have also suggested the role of anion exchangers in the transport of sulfate and Cl- in bovine trachea and human bronchial cells (36). Additionally, it is possible that the airway cation and anion exchangers may be important in maintaining not only the intracellular environment of the epithelial cells but also the pH of the airway surface liquid (ASL; see Ref. 43).

To date, however, the molecular identity of the human bronchial NHE and AE isoforms and their roles in normal physiology and pathophysiology of the human lung are not known. In this report, we investigated the presence of mRNA(s) for various members of the human NHE (Na+/H+ exchangers) and AE (Cl-/HCO-3 exchangers) gene families in various regions of the human conducting airways. Our results demonstrate that the NHE1 isoform of the NHE gene family is expressed in various regions of the proximal and distal human airways, whereas NHE2 and NHE3 mRNA could not be detected. Among the members of the AE gene family, AE2 and brain AE3 (bAE3) mRNAs were present throughout the human airways, whereas the mRNA for the human AE1 and cardiac AE3 (cAE3) isoforms could not be detected. The relative abundance of the mRNA for the human NHE1 was higher in the trachea compared with that in distal airways, whereas the abundance of the mRNA for the AE2 isoform remained unchanged in proximal and distal airways.


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INTRODUCTION
METHODS
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Harvesting of the Bronchial Epithelial Cells

Because ion transport characteristics in a number of organs, e.g., kidney and intestine, exhibit marked regional variations, epithelial cells were collected from various regions of the human bronchial tree. Whole human lungs collected from organ donors were packed on ice and transported to the laboratory. Organ donor lungs were provided by the Regional Organ Donor Bank of Illinois. The time elapsed between lung resection and arrival to the laboratory was between 4 and 6 h. The lung was placed on a waxed dissection tray, and the airway lumen was opened longitudinally and rinsed with Krebs-Henseleit solution. The mucosa was then gently scraped with a scalpel, and epithelial pellets from each bronchial region were immediately placed in separate vials containing chilled RNazol reagent (Tel-Test, Friendswood, TX) and were transferred to a -80°C freezer. Bronchial regions were designated broadly as large (1st to 3rd generation), medium (3rd to 7th generation), and small bronchi (7th to 9th generation). The average age of the donors was 45 ± 4.2 yr, and of the 19 donors obtained over the course of studies, 14 were men and 5 were women. These investigations were approved by the Institutional Review Board of the University of Illinois at Chicago and by the Human Investigations Subcommittee of the West Side Veterans Affairs Medical Center.

RT-PCR and RNase Protection Assay Methods

Designing of PCR primer sets for NHE1, NHE2, NHE3, AE1, AE2, bAE3, and cAE3 and glyceraldehyde-3-phosphate dehydrogenase. The gene-specific PCR primer sets for the human NHE1, NHE2, NHE3, AE1, AE2, bAE3, and cAE3 were designed to detect the isoform-specific mRNA in the human airways. The sequence information of these isoforms for human NHE1, NHE3, AE1, AE2, bAE3, and cAE3 was retrieved from the GenBank CD-ROM supplied with Gene Works software. Human NHE2 primers were designed from the partial cDNA sequence for this isoform cloned in our laboratory (13): NHE1: 5' primer, 5'-CCAGCTCATTGCCTTCTACC-3' and 3' primer, 5'-TGTGTCTGTTGTAGGACCGC-3' [length of amplified region 245 residues; nucleotides (nt) 1766-2010 of the human NHE1, M81768 (35)]; NHE2: 5' primer, 5'-GAAGATGTTTGTGGACATTGGGG-3' and 3' primer, 5'-CGTCTGAGTCGCTGCTATTGC-3' [length of amplified region 550 residues; nt 1633-2182 of the human NHE2 clone, AF073299 (J. Malakooti, R. Y. Dahdal, L. Schmidt, P. K. Dudeja, T. J. Layden, and K. Ramaswany, unpublished results), corresponding to nt 1744-2293 of the rat NHE2, L11004 (12)]; NHE3: 5' primer, 5'-GCAGACCTGGCTTCTGAACC-3' and 3' primer, 5'-GGAACTTCCTGTCGAAGTGG-3' [length of amplified region 380 residues; nt 1174-1553 of the human NHE3, U28043 (9)]; AE1: 5' primer, 5'-CCAGACTCCAGCTTCTACAAGG-3' and 3' primer, 5'-GGAAGGAGAAGATCTCCTGG-3' [length of amplified region 520 residues; nt 1174-1693 of the human AE1, M27819 (21)]; AE2: 5' primer, 5'-GAAGATTCCTGAGAATGCCG-3' and 3' primer, 5'-GTCCATGTTGGCACTACTCG-3' [length of amplified region 181 residues; nt 1698-1878 of the human AE2, U62531 (22)]; bAE3: 5' primer, 5'-ATCTGAGGCAGAACCTGTGG-3' and 3' primer, 5'-TTTCACTAAGTGTCGCCGC-3' [length of amplified region 418 residues; nt 452-869 of the human bAE3, U05596 (44)]; cAE3: 5' primer, 5'-TTTGAGGATGGTGACCTGTG-3' and 3' primer, 5'-CTTGTCATCGTTGGGATGG-3' [length of amplified region 552 residues; nt 260-811 of the human cAE3, U05597 (44)].

Synthesis of oligonucleotides and sequence analysis. Nucleic acid and protein sequence analysis was performed with GeneWorks software (Intelli-Genetics, Mountain View, CA) on a Power Macintosh 6100/60 computer. GenBank and other sequences were retrieved from the CD-ROM supplied with GeneWorks software. Oligonucleotides were obtained from Midland Certified Reagent (Midland, Texas).

Isolation of RNA. Total RNA was extracted from various tissue sources by the method of Chomczynski and Sacchi (10) using RNazol solution supplied by the manufacturer (Tel-Test) and essentially using the manufacturer's protocol.

RT-PCR technique. RT-PCR was performed essentially as described (31, 34). Briefly, 5-10 µg of total RNA were used for RT with gene-specific primers and Superscript II RT enzyme utilizing the Superscript kit for first-strand cDNA synthesis from GIBCO BRL (Gaithersburg, MD). The reaction was carried out in a 25-µl reaction containing 100 mM Tris · HCl, pH 8.3, 10 mM MgCl2, 10 mM dithiothreitol, 50 mM KCl, 1 mM deoxynucleotide triphosphates, 50 µg/ml actinomycin D, 4 µM of antisense primer, and 10 units of RT enzyme and was incubated at 42°C for 1 h. After the RT enzyme reaction, samples were precipitated with ethanol before the PCR reaction. The PCR reaction was carried out using standard step-cycling conditions with 30 cycles of amplification utilizing Taq DNA polymerase (Perkin-Elmer, Norwalk, CT). The cycling conditions for NHE1, NHE2, and NHE3 were 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min; for AE1, bAE3, and cAE3 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min; and for AE2 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min; all PCRs with performed with 3 mM MgCl2. RT-PCR products were separated by electrophoresis on an 1% agarose gel containing ethidium bromide (0.5 µg/ml). Bands of expected sizes were visualized under ultraviolet light.

Generation of cRNA probes. RT-PCR products for NHE1 were digested with Bgl II and Pst I, and the resulting fragments of ~160 base pairs (bp) were ligated in BamH I and Pst I sites of pGEM 4Z vector (Promega, Madison, WI). NHE1 vectors were then linearized by digestion with Sma I or EcoR I and transcribed with T7 RNA polymerase (Promega). [32P]cRNA contained 210 bp, and the protected fragment corresponded to 160 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RT-PCR products were digested with Nco I and BspH I, and the 210-bp Nco I and BspH I fragment was inserted in the Nco I site of pGEM 5Z vector (Promega). GAPDH vectors were then linearized by digestion with Nco I and transcribed with SP6 RNA polymerase (Promega). [32P]cRNA for GAPDH contained 300 bp, and the predicted protected fragment corresponded to 210 bp. RT-PCR products for AE2 (181 bp) were ligated into the PCRII vector utilizing the TA cloning kit (Invitrogen, Carlsbad, CA). AE2 vectors were linearized by digestion with BamH I and transcribed with T7 RNA polymerase (Promega). [32P]cRNA contained 290 bp, and the protected fragment corresponded to 181 bp.

Sequencing of RT-PCR products. RT-PCR products cloned into pGEM vectors or into PCRII vectors were sequenced with universal primer and reverse sequencing primer for M13/pUC, which were purchased from New England Biolabs (Beverly, MA). Sequencing reactions were conducted essentially following the protocol supplied by the manufacturer for the Sequenase kit (USB, Cleveland, OH). Sequence ladders were run on a polyacrylamide gel cast on a Sequi-Gen Nucleic Acid Sequencing System (Bio-Rad, Richmond, CA).

RNase protection assay. For quantitation of mRNA level, the RNase protection assay was utilized due to its high sensitivity and feasibility with small quantities of RNA isolated from scraped cells of even distal airways. 32P-labeled antisense riboprobes were transcribed from either T7 or SP6 promoter in the presence of [alpha -32P]CTP using the Riboprobe Gemini transcription system (Promega). The RNase protection assay was performed as previously described (14, 34). Briefly, total RNA (10-20 µg) was coprecipitated with 105 counts/min of the 32P-labeled probe. Samples were then resuspended in a hybridization buffer containing 75% formamide, 400 mM NaCl, 1 mM EDTA, and 40 mM PIPES, pH 6.4, and hybridized at 48°C for 12-18 h. Samples were then diluted in 10 volumes of 300 mM NaCl, 5 mM EDTA, and 10 mM Tris, pH 7.5, and 1,400 units of T1 ribonuclease were added to each sample. After a 45-min incubation at 37°C, samples were added to a stop solution containing 4 M LiCl and 5 µg of tRNA and precipitated with 2 volumes of ethanol. Precipitates were resuspended in a small volume of dye solution (xylene cyanole and bromphenol blue in 90% formamide and 10 mM EDTA, pH 7.5). The double-stranded [32P]RNA fragments that were protected from RNase digestion were heated at 95°C for 5 min and analyzed by electrophoresis on a denaturing polyacrylamide gel containing 8 M urea. The gels were dried and exposed to Kodak X-AR5 film. The resulting autoradiograms were scanned on a model RC 3079 Ephortec scanning densitometer (Joyce Loebl, Gateshead, UK). The films were exposed to radioactive gels for varying lengths of time to ensure that the signal densities were within the narrow linear range of the film. Furthermore, because the GAPDH mRNA was expressed in very high abundance compared with NHE or AE isoforms, during the synthesis of 32P-labeled cRNA probe for GAPDH, GAPDH probes were synthesized utilizing higher template amounts of cold GAPDH compared with NHE1 or AE2 (ratio of 5:1). Also, cold CTP ratio to 32P-labeled CTP was 4:1 for GAPDH versus NHE1 or AE2. This was specifically done to synthesize a lower specific activity 32P-labeled GAPDH probe compared with NHE1 or AE2 to keep the density of GAPDH in a readable range on final autoradiograms while still enabling the detection of NHE1 and AE2 isoforms. This procedure of labeling was exactly the same each time, and once the probes were synthesized, the same batch of probes was simultaneously utilized for hybridization with equal amounts of RNA extracted from small, medium, and large bronchi and trachea.

Statistical Analysis

All experiments were performed utilizing three or four independent sets of organ donor lung specimens, each from separate individuals. Results are expressed as means ± SE. Student's t-test was used in statistical analysis. A P value of <0.05 was considered significant.


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Detection of the Human NHE Isoforms

RT-PCR assays were performed to detect the presence of human NHE1, NHE2, and NHE3 mRNAs in various regions of the human airways. The PCR primer pair sequences for NHE1, NHE2, and NHE3 are given in METHODS. Initially, RT-PCR was performed with total RNA extracted from human lung small, medium, and large bronchial and tracheal cells using gene-specific primers for the human NHE1, NHE2, and NHE3. As shown in Fig. 1A, PCR products of the expected size corresponding to 249 bp for NHE1 were detected in all regions of the human airways, and the mRNAs for NHE2 (550 bp) and for NHE3 (380 bp) could not be detected in various regions of the airways (Fig. 1A). A negative control was used to rule out any contamination due to genomic DNA, with RT reactions run in the absence of the RT enzyme followed by PCR (Fig. 1B). Additionally, the absence of NHE mRNAs was further confirmed by appropriate positive controls, e.g., simultaneously running RT-PCR with the RNA from the human colon, which exhibited synthesis of the PCR product for both the NHE2 and NHE3 isoforms. As shown in Fig. 2A, utilizing gene-specific primers for NHE3, an expected size fragment of 380 nt was synthesized only in the human descending colon (lane B), whereas no NHE3 PCR products were synthesized with the RNA from small, medium, and large bronchial and tracheal regions (lanes C-F). Similar results were obtained for the NHE2 isoform (Fig. 2B), where, utilizing gene-specific primers for NHE2, an expected size fragment of 550 bp was synthesized only in the human descending colon (lane B), whereas no NHE3 PCR products were synthesized with the RNA from small, medium, and large bronchial and tracheal regions (lanes C-F). The NHE1 specific PCR products were excised and eluted from the agarose gel. The eluted PCR fragments were digested with Pst I and Bgl II and ligated on the BamH I and Pst I cut pGEM 4Z vector. This cloned PCR fragment showed 100% sequence homology with the published human NHE1 sequence (35).



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Fig. 1.   A: RT-PCR analysis of Na+/H+ exchanger (NHE) isoform 1 (NHE1), NHE2, and NHE3 message in human bronchial regions. Lanes A and K: DNA ladder 123; lanes B-D: NHE1, NHE2, and NHE3, respectively, for small bronchi; lanes E-G: NHE1, NHE2, and NHE3, respectively, for medium bronchi; lanes H-J: NHE1, NHE2, and NHE3, respectively, for large bronchi. B: negative controls for RT-PCR analysis of NHE1 mRNA in human bronchial regions. Lanes A-C: NHE1 in small, medium, and large bronchial regions, respectively; lanes D-F: RT-PCR with NHE1 primers in the absence of RT enzyme in RT reaction for small, medium, and large bronchial regions, respectively.




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Fig. 2.   RT-PCR analysis of NHE3 message in human descending colon and bronchial regions. Lane A: DNA ladder 123; lane B: NHE3 for human descending colon (positive control); lanes C-F, NHE3 for small, medium, and large bronchial regions and trachea, respectively. B: RT-PCR analysis of NHE2 message in human descending colon and bronchial regions. Lane A: DNA ladder 123; lane B, NHE2 for human descending colon (positive control); lanes C-F: NHE2 for small, medium, and large bronchial regions and trachea, respectively.

Detection of Human AE Isoforms

RT-PCR assays were also performed to detect the presence of human AE1, AE2, bAE3, and cAE3 mRNAs in various regions of the human airways. Initially, RT-PCR was performed with total RNA isolated from bronchial (small, medium, large) and tracheal cells of the human lung using gene-specific primers for the human AE1, AE2, bAE3, and cAE3. As shown in Fig. 3, the PCR product of the expected size of AE2 (182 bp) could be synthesized from the total RNA from the small, medium, and large bronchi (lanes E, I, and M, respectively), whereas the mRNA for AE1 (520 bp) could not be detected in all three regions (lanes C, G, and K). As also shown in Fig. 3, negative controls with RT enzyme omitted from the RT reactions failed to synthesize the expected size product for AE1 and AE2, confirming that the detection of the AE2 mRNA in various regions of the human airways was not due to contamination by genomic DNA. As shown in Fig. 4, products of the expected sizes corresponding to 418 bp for bAE3 were detected in all regions of the human airways as well as in the positive control, the human brain RNA (Clontech, Palo Alto, CA). The mRNA for cAE3 (551 bp) was found to be absent in various regions of the airways but could be synthesized from the positive control, the human cardiac RNA (Clontech). The AE2- and bAE3-specific PCR products were excised and eluted from the agarose gel. The eluted PCR fragments were cloned in PCRII vector utilizing the TA cloning kit (Invitrogen, San Diego, CA). These cloned PCR fragments showed 100% sequence homology with the published human AE2 and bAE3 sequences (11, 44).


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Fig. 3.   Negative controls for RT-PCR analysis of Cl-/HCO-3 exchanger (AE) isoform 1 (AE1) and AE2 message in human bronchial regions. RT-PCR was performed in the presence (+RT) or absence (-RT) of RT enzyme. Lane A: DNA ladder 123; lanes B-E, small bronchi AE1 primers (-RT) (B), AE2 primers (-RT) (C), AE1 primers (+RT) (D), and AE2 primers (+RT) (E); lanes F-I: medium bronchi AE1 primers (-RT) (F), AE2 primers (-RT) (G), AE1 primers (+RT) (H), and AE2 primers (+RT) (I); lanes J-M: large bronchi AE1 primers (-RT) (J), AE2 primers (-RT) (K), AE1 primers (+RT) (L), and AE2 primers (+RT) (M).



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Fig. 4.   RT-PCR analysis of brain AE3 (bAE3) and cardiac AE3 (cAE3) message in human airway regions. Lanes A and L: DNA ladder 123; lane B: bAE3 for brain control RNA; lane K: cAE3 for cardiac control RNA; lanes C-F, bAE3 for small, medium, and large bronchial and tracheal RNA, respectively; lanes G-J, cAE3 for small, medium, and large bronchial and tracheal RNA, respectively.

Regional Distribution of the Human NHE1 and AE2 Messages in the Human Proximal and Distal Airways as Assessed by RNase Protection Assay

When [32P]cRNA probes were hybridized to total RNA and subsequently digested, bands of predicted sizes were observed in a quantitative manner. Human GAPDH was used as an internal control. Figure 5 shows a representative RNase protection blot for NHE1 and GAPDH (Fig. 5A) and for AE2 and GAPDH (Fig. 5B) in the human bronchial regions. These figures demonstrate protected fragments for NHE1 and GAPDH of the appropriate expected sizes (Fig. 5A) as well as for AE2 and GAPDH (Fig. 5B) in all regions of the airways. These data again confirm the presence of NHE1 and AE2 mRNAs throughout the human airways.



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Fig. 5.   A: representative RNase protection autoradiogram for NHE1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lanes A and B: undigested probes for GAPDH and NHE1, respectively; lane C: protected GAPDH fragment alone; lane D: tRNA control hybridized to NHE1 and GAPDH probe and digested with RNase; lanes E-H, protected fragments for NHE1 and GAPDH (indicated by arrows) after hybridization to total RNA and digestion with RNase from small, medium, and large bronchi and trachea, respectively. B: representative RNase protection autoradiogram for AE2 and GAPDH. Lanes A and B: undigested probes for GAPDH and AE2, respectively; lanes C and D: protected fragments for GAPDH and AE2 alone, respectively; lane E: tRNA control hybridized to AE2 and GAPDH probe and digested with RNase; lanes F-I, protected fragments for AE2 and GAPDH (indicated by arrows) after hybridization to total RNA and digestion with RNase from small, medium, and large bronchi and trachea, respectively.

Analyses of the RNase protection assays for NHE1 and AE2 along the proximal and distal human airways are depicted in Fig. 6, A and B, respectively. The relative abundance of the NHE1 and AE2 mRNAs (y-axis of Fig. 6) was calculated by taking a ratio of their respective densities to that of GAPDH (internal standard). As shown in Fig. 6B, there were no significant regional differences observed in the mRNA levels of AE2 along the proximal and distal human airways (P > 0.05). In contrast, there were significant regional differences observed in the mRNA levels of NHE1 (when normalized to GAPDH) along the regions of the human airways (Fig. 6A). As can be seen in Fig. 6A, the NHE1 mRNA levels were significantly higher in the trachea compared with those in the distal bronchial regions as analyzed by Student's t-test (P < 0.05). NHE1 mRNA levels were the highest in the trachea followed by large > medium > small bronchi.


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Fig. 6.   Relative abundance of NHE1 and AE2 in the human airways. Relative abundance of NHE1 (A) and AE2 (B) was determined (y-axis) by calculating ratio of their density to density of GAPDH (internal standard). Values are means ± SE of 3-5 independent samples from 3-5 organ donors. Bronch, bronchi.


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NHEs and AEs are nearly ubiquitous plasma membrane transport proteins implicated in the maintenance of intracellular pH and concentrations of intracellular Na+, H+, Cl-, and HCO-3 as well as their vectorial transport (2, 39, 45). Although a number of recent studies have identified the presence of activities of these transporters in either cultured alveolar epithelial cells or tracheal or nasal epithelium (23, 24, 28, 29, 37), our current studies for the first time identify the molecular isoforms of these transporters in the human airways and specifically their distribution in both the proximal and distal airways.

Our data clearly demonstrate that the mRNA for the NHE1 isoform of the NHE gene family is present in proximal as well as distal airways, whereas the mRNA for the human NHE2 and NHE3 genes could not be detected. Furthermore, the mRNAs for the AE2 and the bAE3 isoforms of the anion exchanger gene family were also detected throughout the proximal and distal airway regions, whereas the mRNA for the AE1 and cAE3 could not be detected in these airway regions. Our study describes gross regional distribution of the NHE and AE isoforms and does not discriminate between specific epithelial versus nonepithelial cell types. It can be argued that lack of detection of AE1, cAE3, or NHE2 and NHE3 (relatively larger-size PCR products) could also be due to partially degraded RNA obtained from organ donor lungs. It should be noted that the lack of detection was not due to degraded RNA because with the use of another primer set for human AE2 [for synthesis of human AE2 fragment 2069-2532, U62531 (22)], even the larger-size fragment of 463 nt could also be synthesized from all regions of the small, medium, and large bronchi as well as from the trachea (data not shown), ruling out the possibility that lack of synthesis of the larger-size PCR fragments could be due to degraded RNA.

In this regard, the NHE1 isoform has previously been shown to be a ubiquitous isoform in nearly all mammalian cells (39). Our current findings of NHE1 mRNA being present in all regions of the human airways are consistent with the ubiquitous nature of this NHE isoform. The NHE1 isoform has previously been suggested to be involved in a number of "housekeeping" functions, including maintenance of intracellular pH and regulation of cell volume and cell proliferation (for reviews, see Refs. 30, 39). The ubiquitous nature of this NHE isoform in the human airways, therefore, appears to be compatible with its role in housekeeping functions. The distribution of this isoform in the human airways and its known localization on the basolateral membrane domain of a variety of polarized epithelial cells indicate that this isoform may not be involved in vectorial Na+ absorption in the human airways. In this regard, an Na+/H+ exchange has been identified by Paradiso (29) in basolateral membranes of polarized normal and cystic fibrosis human nasal epithelium. Tessier et al. (37) also provided evidence for the presence of Na+/H+ exchange activity in equine trachea. Acevedo and Steele (1) used ethylisopropyl amiloride and the pH stat method and concluded that sheep tracheal epithelium also possessed an apical membrane Na+/H+ exchange process. In rat type II alveolar epithelial cells, Nord et al. (23, 24) presented evidence for the presence of Na+/H+ exchange. Later, Lubman et al. (19, 20) reported that the Na+/H+ exchange activity was localized to the basolateral side of the epithelial cells grown as monolayers.

In contrast to NHE1, the NHE2 and NHE3 isoforms have previously been shown to be more restricted in their tissue distribution (38, 39). For example, in the rabbit, NHE3 mRNA was detected in the kidney, intestine, and stomach (38). Similarly, in the rat, the NHE3 mRNA has been shown to be detected mainly in the intestine, kidney, and stomach but was also detected in the heart and brain (26). On the basis of tissue distribution studies and localization of this isoform protein to only the apical and not the basolateral membranes, this isoform was suggested to be the most likely candidate involved in neutral NaCl absorption (7, 38, 39). The NHE2 isoform mRNA in the rat and rabbit has also been shown to be predominantly restricted to the stomach, uterus, kidney, intestine, and adrenal glands and much less in trachea and skeletal muscle (41, 45). NHE2 has been shown to be localized to apical membranes of polarized intestinal epithelial cells (16). The absence of both of these putative apical membrane isoforms, NHE2 and NHE3, in the human proximal and distal airways indicates that the neutral NaCl absorptive process (involving dual ion exchange of Na+/H+ and Cl-/HCO-3) may be absent in the luminal membranes of the human airway epithelial cells. Further studies are required to confirm this conclusion because another NHE isoform may be localized to luminal membranes of airways. The NHE1 isoform expressed in the airway epithelial cells may play an important role in a series of basic cellular functions, e.g., maintenance of intracellular pH, cell volume, and cell proliferation.

AE2 and bAE3 are the anion exchanger isoforms expressed in the human airways. In this regard, AE2 cDNA has been cloned from the mouse, rat, rabbit, and human, and the mRNA for this isoform has been shown to be widely distributed in epithelial and mesenchymal tissues (2). Nozik-Grayck et al. (25) demonstrated the presence of AE2 polypeptide in rabbit lung alveoli and medium airway epithelial cells. AE2 polypeptide has been reported to be localized to the basolateral membranes of the alveolar epithelial cell monolayers (20), choroid plexus epithelium, and gastric parietal cells in the human (2) and rat kidney (3). In contrast, Chow et al. (11) reported that AE2 was localized to the apical membranes and not the basolateral membranes in the rabbit ileum. Rossman et al. (33), however, demonstrated that AE2 was localized to the basolateral and not the apical membranes in both the rabbit ileum and stomach. Recent studies from our laboratory have also demonstrated that AE2 and bAE3 polypeptides were predominantly localized to the basolateral membranes in the human colon and ileum (4). The bAE3 and cAE3 are the alternatively spliced isoforms cloned from the human heart, and their tissue and membrane distributions have not been investigated in detail (2, 44). In this regard, Tessier et al. (37) also provided evidence for the presence of a Cl-/HCO-3 exchange activity in equine trachea. Nord et al. (24) presented evidence for the presence of a DIDS-sensitive and Na+-independent Cl-/HCO-3 exchange process in the rat type II alveolar epithelial cells. This activity was shown to be localized to the basolateral side of the alveolar epithelial cells grown as monolayers (20). These anion exchangers may be involved in the maintenance of intracellular pH, Cl- concentration, volume regulation, and the CO2 excretory function of lungs. The cellular and membrane localization and detailed functional roles of the AE2 and bAE3 isoforms expressed in the human airways remain to be defined.

Additionally, it is reasonable to speculate about the role of anion and cation exchangers in maintaining not only the intracellular pH of the airway epithelial cells but possibly also the pH of ASL. For example, in ferret trachea, the pH of ASL is acidic (pH 6.85-7.12) compared with submucosal solution (pH 7.39) and is maintained relatively constant despite large alterations in submucosal solution (18, 32). In this regard, homeostasis of ASL pH and hence the overlying mucous layer pH are important in maintaining the optimal function of mucociliary clearance mechanisms. Lowering of the mucus pH increases mucus viscosity (6) due to an increase in cross-linkage between mucin macromolecules by H+ (15) and hence could hamper ciliary clearance. Conversely, a rise in mucosal pH increases bacterial binding to tracheal cells (27) and, therefore, might lead to enhanced local bacterial growth and tracheobronchial infection.

In summary, our current studies demonstrate for the first time the expression of the mRNAs for NHE1 and AE2 as well as for bAE3 of the NHE and AE gene families along the proximal and distal human airways. Future studies of these isoforms with respect to their cellular and membrane localization and their functional significance would be of great significance to further define the role of these transporters in normal physiology and pathophysiology of the human airways.


    ACKNOWLEDGEMENTS

We are indebted to the staff of the Regional Organ Bank of Illinois for their dedication and help.


    FOOTNOTES

These studies were supported by a Merit Review Award (P. K. Dudeja) from the Dept. of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases funds.

Preliminary reports of this work have been presented as abstracts (J. Invest. Med. 43: 276A, 1995; FASEB J. 9: A278, 1995; and FASEB J. 10: A357, 1996).

Address for reprint requests and other correspondence: P. K. Dudeja, Univ. of Illinois at Chicago, Medical Research Service (600/151), VA Medical Center, 820 South Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.edu).

Received 27 October 1998; accepted in final form 19 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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