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Am J Physiol Cell Physiol 293: C1814-C1823, 2007. First published October 3, 2007; doi:10.1152/ajpcell.00358.2007
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments

Jia-Jiun Yan,1,2 Ming-Yi Chou,1,3 Toyoji Kaneko,4 and Pung-Pung Hwang1

1Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan; 2Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan; 3Institute of Fishery Science, National Taiwan University, Taipei, Taiwan, Republic of China; and 4Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan

Submitted 10 August 2007 ; accepted in final form 2 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian nephrons, most of the Na+ and HCO3– is reabsorbed by proximal tubular cells in which the Na+/H+ exchanger 3 (NHE3) is the major player. The roles of NHEs in Na+ uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish (Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1–8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H+-ATPase but not with Na+-K+-ATPase, indicating that H+-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na+ FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H+-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H+-ATPase are probably involved in Na+ uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.

ion uptake; acid-base regulation

IN MAMMALIAN NEPHRONS, about 70% of sodium and 80% of bicarbonate are reabsorbed from the lumen by active transport in proximal tubules (38), and the major apical plasma membrane Na+ transporter in the proximal tubular cells is the Na+/H+ exchanger (NHE, of the SLC9 family) (6). Several isoforms, NHE2, -3, and -8, have been localized in the brush-border membrane of proximal tubules, and about 50% of the overall apical NHE activity is mediated by NHE3 (8). Up to 40% of proximal tubular bicarbonate reabsorption is Na+ independent and sensitive to V-type H+-ATPase (HA) (7, 41). According to the current model, HA works in coordination with carbonic anhydrases (CAs) to provide the driving force for the operation of NHE (38).

When compared with terrestrial animals, fish have to cope with more challenging osmotic and ionic gradients from aquatic environments with diverse salinities, ion compositions, and pH values. Similar to mammalian kidneys, gills in freshwater (FW) teleosts are the main organ responsible for Na+ absorption and acid-base regulation (15). So far, two pathways for these mechanisms have been proposed in fish gill ionocytes: 1) an apical HA electrically linked to the Na+ absorption via the epithelial Na+ channel (ENaC) and 2) an electroneutral exchange of Na+ and H+ via proteins of the NHE family; these two pathways are still being debated among different species and various external water conditions, such as salinity and pH (9, 15, 28). Immunocytochemical, pharmacological, and molecular physiological studies have demonstrated the role of HA in Na+ absorption/acid-base regulation mechanisms in FW fish gill cells; however, convincing molecular evidence for the existence of an ENaC in fish gill cells is still unavailable (14, 15, 18, 19, 27, 32, 35).

The uptake of Na+ via passive exchange with H+ in fish gill cells has been questioned on thermodynamic grounds (2, 15, 24). According to the model of mammalian proximal tubular cells, NHE2 and -3 are expected to be the target transporters involved in apical Na+ uptake functions in FW fish gill cells (13, 31). With the use of heterologous antibodies, NHE2 and -3 immunoreactivities were located in gill mitochondrion-rich (MR) cells of several species (13, 42), and homologous antibodies and molecular probes were recently used to provide convincing evidence for the expression of NHE3 in the apical membrane of gill MR cells in a unique FW teleost, the Osorezan dace (Tribolodon hakonensis) (17). Obviously, it is critically important to determine the specific isoforms existing in fish gill ionocytes before we study the roles of NHE in fish gill Na+ absorption/acid-base regulation mechanisms. Zebrafish (Danio rerio), with an extensive genomic database, provides an excellent model to determine the target NHE isoforms. Indeed, recent inhibitor experiments demonstrated the inhibitory effects of 100 µM amiloride and/or 10 µM 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on zebrafish Na+ uptake (4, 12), implying the possible involvements of both ENaC and NHE in zebrafish gill/skin Na+ uptake mechanisms (20).

In the acid-tolerant fish, the Osorezan dace, Hirata et al. (17) suggested that NHE3, not HA, plays the major role in acid secretion in gill MR cells during acclimation to acidic FW. On the other hand, amiloride and EIPA, which inhibit Na+ uptake, had no effect on net acid secretion in FW rainbow trout, brown trout, or European flounder (25, 29, 30, 34, 44). Boisen et al. (4) reported that responses of Na+ uptake to several inhibitors, including bafilomycin, ethoxzolamide, and amiloride, were variable in zebrafish acclimated to soft water and hard water. Craig et al. (12) also reported that acclimation to soft water and hard water caused differential regulations of the mRNA expressions of HA and NHE2 in zebrafish gills. Taking all of these findings into consideration, both NHE and HA/ENaC are probably involved in Na+ uptake/acid-base regulation in fish gills, but the partitions of these two pathways may be modulated depending on the environment, as Hwang and Lee (20) proposed. The present study attempted to test this hypothesis. Zebrafish were used as the model organism to examine the effects of environmental conditions (low Na+ or acidic) on the expressions of nhe and atp6v0c gill cells. In the present study, 1) cDNAs of the nhe gene family in zebrafish (znhe) were cloned and sequenced; 2) expression patterns of znhes in various tissues of zebrafish were examined; 3) cellular localization of NHEs (and znhe mRNAs), HA, and Na+-K+-ATPase (NKA; a marker for MR cells) in zebrafish gills was conducted; and 4) mRNA expressions of znhe and atp6v0c in gills were compared between zebrafish acclimated to 10 and 0.04 meq/l Na+ or pH 6.8 and 4 FW.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental animals. The AB strain of zebrafish, obtained from stocks of the Institute of Cellular and Organismic Biology, Academia Sinica, was kept in a circulating system at 28.5°C under a 14:10-h light-dark photoperiod. Fish were fed artificially bred brine shrimp but were starved during the acclimation experiments (see below). The experimental protocols were approved by the Academia Sinica Institutional Animal Care and Utilization Committee (approval no. RFiZOOHP2006040).

Acclimation experiments. High-Na+ (10 meq/l) and low-Na+ (0.04 meq/l) artificial FW were prepared with double-deionized water (Milli-RO60; Millipore) added with adequate CaSO4·2H2O, MgSO4·7H2O, NaCl, K2HPO4, and KH2PO4. Except for Na+, other ion concentrations and the pH of the media were the same (Table 1). Local tap water (control, pH 6.7–6.9) and acidic FW (pH 4.00–4.05) were also prepared to determine the effects of an acidic medium. The acidic medium was made by adding H2SO4 to local tap water, and the concentrations of other ions in the acidic FW were maintained the same as that in the control (local tap water) (Table 1). Adult zebrafish were acclimated for 7 days to high-Na+, low-Na+, and acid FW and local tap water, and all showed normal swimming behavior with no mortality during the acclimation period. During the experiments, high-Na+ and low-Na+ FW were replaced every 2 days to maintain the proper ion concentrations, and prepared acidic FW stock was continuously pumped into the experimental tank bottom with an electrical pump to maintain a stable pH. All experimental media were checked for pH with a pH meter (MP225; Mettler-Toledo, Schwerzenbach, Switzerland) and for ion concentrations with an atomic absorption spectrometer (U-2000; Hitachi, Tokyo, Japan).


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  		Table 1. Ionic compositions in experimental media

 
Cloning of zNHE. mRNA was purified from the total RNA of zebrafish tissues with a commercial kit (Oligitex; Qiagen, Hilden, Germany). The cDNA for cloning and RACE (rapid amplification of cDNA ends) was produced using a SuperScript III reverse transcriptase kit (Invitrogen, Carlsbad, CA) and a SMART RACE cDNA amplification kit (Clontec, Mountain View, CA) following the manufacturer's protocols, respectively. For polymerase chain reaction (PCR) amplification, 2 µl of cDNA were used as the template in a 50-µl final reaction volume containing 0.25 mM dNTP, 2.5 units of EX-Taq polymerase (Takara, Shiga, Japan), and 0.2 µM of each primer. The primer sets were obtained from a bioinformatics method as listed in Table 2. PCR products obtained were subcloned into a pGEM-T Easy vector (Promega, Madison, WI), and the amplicons were sequenced to confirm the PCR products. The specific primers of 5' and 3' RACE (Table 3) were designed from the partial sequences obtained from the PCR of the primer sets shown in Table 2. The RACE PCR program followed the manufacturer's protocol, and RACE PCR products were also subcloned into the pGEM-T Easy vector and sequenced.


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  		Table 2. Primer sets for cloning of nhe gene family

 

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  		Table 3. Primer sets for RACE of nhe gene family

 
RT-PCR analysis. For cDNA synthesis, 5 µg of total mRNA were reverse-transcribed in a final volume of 20 µl containing 0.5 mM dNTPs, 2.5 µM oligo(dT)18, 5 mM dithiothreitol, and 200 units of PowerScript reverse transcriptase (Invitrogen) for 1.5 h at 42°C and followed by a 15-min incubation at 70°C. For PCR amplification, 1 µl of cDNA was used as a template in a 25-µl final reaction volume containing 0.25 µM dNTP, 1.25 units of Gen-Taq polymerase (Genemark, Taipei, Taiwan), and 0.2 µM of each primer.

Phylogenetic tree analysis. Full-length cDNAs were obtained after the RACE PCR and sequenced. The entire amino acid sequences deduced with the CLUSTAL program were used for the multiple sequence alignment and phylogenetic tree analysis (16). The data sets were treated by the neighbor-joining (NJ) analysis method, and 1,000 bootstrap replicates of analysis were carried out with the MEGA program (version 3.1).

Quantitative RT-PCR. Quantitative RT-PCR (qRT-PCR) was performed with an ABI7000 sequence detection system (ABI, Warrington, UK) in a final volume of 10 µl, containing 5 µl of 2x SYBR green master mix (ABI), 50 nM of the primers pairs, and 3.2 ng of cDNA. The standard curve of each gene was checked in a linear range with β-actin as an internal control. The primer sets for the qRT-PCR are shown in Table 4.


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  		Table 4. Primer sets for quantitative RT-PCR and tissue distribution

 
In situ hybridization. Different nhe probe fragments were obtained by PCR and inserted into a pGEM-T easy vector (Promega). After PCR with the T7 and SP6 primers, the products were subjected to in vitro transcription with T7 and SP6 RNA polymerase (Roche, Penzberg, Germany), respectively. Digoxigenin (DIG)-labeled RNA probes were examined with RNA gels to confirm the quality and concentration. Gills collected from zebrafish were fixed in a 4% paraformaldehyde (PFA) phosphate-buffered saline (PBS; 1.4 mM NaCl, 0.2 mM KCl, 0.1 mM Na2HPO4, and 0.002 mM KH2PO4) solution at 4°C overnight and then treated with methanol at 4°C overnight again. Afterward, the samples were treated with a methanol series with concentrations from 100 to 25% in diethylpyrocarbonate-PBST (PBS with 0.1% Tween 20) for 10 min each. Samples were then hybridized with the prepared probe in hybridization buffer (60% formamide, 5x SSC, 0.1% Tween 20, 500 µg/ml yeast tRNA, and 50 µg/ml heparin) overnight at 65°C. The next day, samples were washed serially with 75% hybridization buffer in 25% 2x SSC at 65°C up to 100% 2x SSC, and finally with 0.2x SSC for 5–10 min six times. Samples were blocked with 5% sheep serum in 2 mg/ml bovine serum albumin (Sigma, St. Louis, MO) at room temperature for 2–4 h and then incubated with an anti-DIG antibody (1:10,000, in blocking solution) at 4°C overnight. After being washed with DEPC-PBST at room temperature for 15 min eight times, staining buffer (0.1 M Tris, pH 9.5, 50 mM MgCl2, 0.1 M NaCl, and 0.1% Tween 20) was subsequently used for 5 min three times. Staining was conducted with the mixture of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in 10 ml of staining buffer at room temperature from 10 min to several hours (depending on the gene) in the dark. The reaction was stopped by PFA and subsequently washed with methanol for several times. Finally, the samples were stored in PBS at 4°C in a dark box until further examination and analysis. Images were obtained with a stereomicroscope (SZX-ILLD100; Olympus, Tokyo, Japan) or an upright microscope (Axioplan 2 imaging; Carl Zeiss, Oberkochen, Germany). Image analysis was conducted with a program of Olympus DP70-BSW or Sport RT-Color (Diagnostics Instruments, Sterling Heights, MI), respectively.

Cryosectioning. Fresh zebrafish gills or in situ hybridized gills were fixed with 4% PFA at 4°C for 3 h and then immersed serially in PBS containing 5, 10, and 20% sucrose for 15 min at room temperature. For double staining with concanavalin A (ConA), the gills were incubated with 0.05 mg/ml ConA-conjugated Texas red (Invitrogen) for 30 min before fixation. Finally, gills were soaked in a mixed PBS solution (OCT compound: 20% sucrose at 1:2) overnight and then embedded with OCT compound embedding medium (Sakura, Tokyo, Japan) at –20°C. Cryosections at 10 µm were made with a cryostat (CM 1900; Leica, Heidelberg, Germany), and these were placed onto poly-L-lysine-coated slides (EMS, Hatfield, PA).

Immunocytochemistry. Prepared slides were rinsed in PBS and blocked with 3% BSA for 30 min. Afterward, the slides were first incubated with an {alpha}5 monoclonal antibody against the {alpha}-subunit of the avian NKA (Hybridoma Bank, University of Iowa, Ames, IA; 1:600 dilution) overnight at 4°C. The slides were washed twice with PBS and incubated with an Alexa Fluor 568 goat anti-mouse IgG antibody (Molecular Probes, Carlsbad, CA; 1:200 diluted with PBS) for 2 h at room temperature. After being washed with PBS twice, the slides were incubated again with a polyclonal antibody against the A-subunit of killifish HA (1:300 diluted with PBS) (22) or with a polyclonal antibody against dace NHE3 (1:16,000 diluted with PBS) (17) overnight at 4°C. Thereafter, the slides were incubated with Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes; diluted 1:200 with PBS) for 2 h at room temperature. For double staining with 4,6-diamidino-2-phenylindole (DAPI), the slides were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Images were acquired with a Leica TCS-NT confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany) or an Axioplan 2 imaging microscope (Carl Zeiss).

Statistical analysis. Values are means ± SD and were compared using Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning, sequencing, and bioinformatics analysis of the zebrafish nhe gene family. Eight members of the znhe family were predicted from the ENSEMBL and NCBI databases. Subsequently, full-length cDNAs of the eight znhe members were cloned and sequenced from zebrafish (Fig. 1 and Supplemental Fig. 1). Each full-length sequence contained 5' and 3' untranslated regions (UTRs) and a 3' UTR with a poly(A) tail. The complete cDNA sequences of the znhes are 2,200–4,500 bp, which contain 2,000- to 2,500-bp coding regions. After blasting the NCBI and ENSEMBL genome databases with the BLASTN tool of Genome Blast, we found znhe3a and znhe3b to be on chromosome 19. Other isoforms, znhe1, -2, -5, -6, -7, and -8, were on chromosomes 2, 9, 7, 10, 6, and 23, respectively. The coding regions of the znhe gene family were translated as 650–970 amino acids and contain 10–13 transmembrane domains.


Figure 1
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  		Fig. 1. Nucleotide and deduced amino acid sequences of nhe3b cDNA from zebrafish (accession no. EF591980). Bold type, sodium affinity site (ratNHE1); open box, intracellular pH-sensitive site (humanNHE1); shaded region, predicted transmembrane domain; underlined type, NHE conserved domain.

 
A phylogenetic tree was generated based on the NJ analysis of the full-length amino acid sequences of the NHE isoforms from human, mouse, rat, trout, and dace. The zNHE isoforms were homologous to their mammalian counterparts. According to the phylogenetic tree (Fig. 2), both zebrafish and human NHE were separated into two subfamilies, NHE1–5 and NHE6–9, respectively. However, there was another new mammalian NHE isoform (named sperm-specific NHE, or NHE10; Ref. 40) that formed an additional branch.


Figure 2
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  		Fig. 2. Phylogenetic tree constructed with the complete amino acid sequences of Na+/H+ exchangers (NHEs). The analysis was conducted using the neighbor-joining method with 1,000 bootstrap replicates. Numbers indicate the bootstrap values. The GenBank accession numbers of the sequences used are as follows: zebrafish (z)NHE1, EF591982; trout β-NHE1, M94581; human (h)NHE1, NP_003038; ratNHE1, NP_036784; zNHE2, EF591983; hNHE4, NP_001011552; mouseNHE4, NP_796058; hNHE2, NP_003039; mouseNHE2, NP_001028461; zNHH3a, EF591984; daceNHE3, BAB83083; hNHE3, NP_004165; mouseNHE3, XP_127434; zNHE5, EF591985; hNHE5, NP_004585; ratNHE5, NP_620213; zNHE8, EF591988; hNHE8, NP_056081; mouseNHE8, NP_683731; zNHE6, EF591986; hNHE6, NP_006350; mouseNHE6, NP_766368; zNHE7, EF591987; hNHE7, NP_115980; mouseNHE7, NP_796327; hNHE9, NP_775924; mouseNHE9, NP_808577; hNHE10, NP_898884; and mouseNHE10, NP_932774. Scale bar represents 20% replacement of amino acids per site.

 
Commercial software (GeneDoc, Pittsburgh, PA) was used to analyze the alignment of the amino acid sequences. The zNHE3a had 64% identity with zNHE3b, and 66% identity was found between zNHE6 and zNHE7. If we aligned the predicted transmembrane domains only, the identities were higher. For example, zNHE3a and zNHE6 showed 78 and 73% identities to zNHE3b and zNHE7, respectively. The zNHEs share relatively high similarities with their mammalian counterparts in the membrane-spanning domains, but they greatly diverge in the intracellular COOH-terminal regions.

Expression patterns of zebrafish nhe family in different tissues. The expressions of nhe mRNAs in different tissues of zebrafish were examined by RT-PCR with β-actin mRNA as an internal control. Figure 3 clearly indicates that znhe1 was primarily expressed in erythrocytes, and znhe2 and znhe3b are the primary isoforms expressed in gills. Furthermore, both znhe3b and znhe3a were also abundantly expressed in kidneys. Small amounts of znhe5 and znhe6 were expressed in the brain and gills. Testis showed abundant expression levels of znhe2 and znhe7. On the other hand, znhe8 was the most ubiquitous isoform that was expressed in all tissues.


Figure 3
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  		Fig. 3. RT-PCR analysis of the expressions of the znhe family in various tissues of zebrafish. β-Actin was used as the internal control. The amplicon sizes of different genes are shown in Table 4.

 
Effects of environmental pH and Na+ concentration on mRNA expressions of the znhe family in gills. As shown in Fig. 3, all znhe members except znhe3a were found (by RT-PCR analysis) to be expressed in zebrafish gills, which is the major organ for Na+ uptake and acid-base regulation. To identify the isoforms that are specifically responsible for these mechanisms, we predicted the expressions of the target isoforms in zebrafish gills to be regulated during environmental acidic or low-Na+ challenges, and therefore the hypothesis was tested in subsequent experiments. As indicated in Fig. 4, only the expression (according to the RT-PCR analysis) of znhe3b appeared to be affected after acclimation to acid, indicating znhe3b is the target isoform relevant to the mechanisms of Na+ uptake/acid-base regulation in zebrafish gills. Based on this finding, the following experiments mainly focused on znhe3b.


Figure 4
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  		Fig. 4. RT-PCR analysis of the expressions of the znhe family in gills of zebrafish acclimated to control fresh water (C) and acidic water (A) for 7 days. β-Actin was used as the internal control. The amplicon sizes of different genes are shown in Table 4.

 
Localization of zNHE3b in zebrafish gill cells. Subsequent immunocytochemical and in situ hybridization experiments demonstrated that mRNA expression of znhe3b was in a specific group of ionocytes in zebrafish. Double immunocytochemical labeling for NHE3b and NKA supported the above RT-PCR results (Fig. 4). In Fig. 5A, NHE3b was immunocytochemically localized in a group of cells in the gills. As shown in Fig. 5B, NKA was expressed in the basolateral membrane of Na+-pump rich (NaR) cells (i.e., MR cells) and was not colocalized with NHE3b (Fig. 5C). Subsequent triple labeling for ConA (an apical marker for HR cells; Ref. 26), DAPI (a marker for nuclei), and NHE3b demonstrated that the signals of ConA and NHE3b were colocalized at the apical opening of the specific group of cells (Fig. 5, E–H), which would later be identified as HR cells in the following in situ hybridization experiments (see below).


Figure 5
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  		Fig. 5. Confocal images of double labeling for NHE3b and NKA proteins (A–D) and triple labeling for 4,6-diamidino-2-phenylindole (DAPI), concanavalin A (ConA), and NHE3b protein (E–H) in zebrafish gill cryosections. A: NHE3b. B: Na+-K+-ATPase (NKA). C: merged image of A and B. D: bright-field image of the filament section. DIC, differential interference contrast. E: DAPI. F: ConA. G: NHE3b. H: merged image of E–G. NHE3b (outlined with a dashed line) was not colocalized with NKA (arrows). ConA and NHE3b signals were colocalized at the apical region of the cell that was labeled with DAPI. Scale, 5 µm.

 
Triple labeling for znhe3b mRNA, NKA, and HA was further conducted to identify the cell types expressing znhe3b (Fig. 6). Whole mount in situ hybridization was used to detect znhe3b mRNA in the cells of zebrafish (Fig. 6A). In zebrafish gills, znhe3b mRNA signals were abundantly observed in the primary filament and base of secondary lamellae; fewer signals were found in the distal portion of secondary lamellae (Fig. 6A). Cryosections of the whole mount in situ hybridized gills were further double labeled with the anti-HA A-subunit (a marker for HR cells) and anti-NKA antibodies (Fig. 6, B–F). In Fig. 6, B–D, HA and NKA were not colocalized, indicating two distinctive ionocytes, HR cells and NaR cells, in zebrafish gills. Triple-labeling images clearly indicated that znhe3b mRNA was expressed in HR cells but never in NaR cells (Fig. 6, E and F), demonstrating that only HR cells express znhe3b. On the other hand, mRNA signals of the other isoforms, znhe1, -2, -3a, -5, -6, -7and -8, were not detectable in zebrafish gill cells (data not shown). All the immunocytochemistry and in situ hybridization experiments were conducted in more than five individuals, and the results were the same.


Figure 6
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  		Fig. 6. Triple in situ hybridization and immunocytochemical analysis of znhe3b, V-type H+-ATPase (HA), and NKA in zebrafish gill cryosections. A: whole mount in situ hybridization of znhe3b mRNA. B: HA. C: NKA. D: merged image of B and C. E: znhe3b signals in a bright-field image. F: merged image of D and E. znhe3b mRNA was colocalized with HA (outlined with a dashed line) but never with NKA (arrows). Scale, 100 µm in Aand 20 µm in B–F.

 
Effects of environmental pH and Na+ concentration on mRNA expressions of transporters and enzymes in gill HR cells. On the basis of the above results, qRT-PCR was further conducted to examine changes in gill transcripts of znhe3b and zatp6v0c (zebrafish H+-ATPase V0c subunit) during acclimation to acid or low-Na+ FW. As shown in Fig. 7, znhe3b mRNA expression was downregulated (94% of the control) after acid treatment, while at the same time, that of zatp6v0c was upregulated by 138% compared with the control. On the other hand, in the case of acclimation to L-Na FW, the transcription of znhe3b increased 157%, but that of zatp6v0c decreased 58%, compared with the high-Na+ FW (Fig. 7). Apparently, expressions of the two genes in zebrafish gill cells are differentially regulated depending on environmental situations.


Figure 7
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  		Fig. 7. Changes in the transcripts (quantitative RT-PCR) of znhe3b and zatp6v0c in gills of zebrafish acclimated to local tap water (control) and acidic (acid), high-Na+ (H-Na), and low-Na+ (L-Na) fresh water. znhe3b was respectively downregulated and upregulated in acid (compared with control) and L-Na (compared with H-Na), whereas zatp6v0c showed a reverse response. Data are means ± SD (n = 4). *P < 0.05, significant difference (Student's t-test) between control and acidic fresh water and between H-Na and L-Na as indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Full-length cDNAs of the eight znhe isoforms (znhe1, -2, -3a, -3b, -5, -6, -7, and -8) were first cloned and sequenced from zebrafish in the present study. The cDNAs of znhe encode 650–970 amino acids, which contain 10–13 putative hydrophobic transmembrane domains and share 20–66% amino acid sequence identities with paralogous genes. Alignment results indicated that the membrane-spanning domains of zNHEs share relatively high similarities with their mammalian counterparts, but the intracellular COOH-terminal regions of zNHE6–9 were quite divergent and much shorter than those of zNHE1–5. The identities of zNHE1–5 with other similar homologous forms from mammals were lower than 55%, but those of the others were higher than 65% (zNHE6) and even 75% (zNHE7 and zNHE8). According to the NJ phylogenetic tree, the zNHEs were clustered into two distinct groups, zNHE1–5 and zNHE6–8, which correspond to the homologs of other vertebrates, respectively. Interestingly, two isoforms, zNHE3a and zNHE3b, were clustered with the group of NHE3. So far, zebrafish is the only species to possess duplicated nhe3 genes (Fig. 2). The phenomena of gene duplication in teleost fish have been extensively studied (1, 21). Similar gene duplication has been reported in other ion transporters. In the case of NKA, mammals have only one {alpha}1-subunit, whereas zebrafish have as many as five different isoforms, {alpha}1a.1, {alpha}1a.2, {alpha}1a.3, {alpha}1a.4, and {alpha}1b (5).

The expression patterns of the mammalian NHE gene family have been extensively studied. NHE1 is ubiquitously expressed in the basolateral plasma membrane; NHE2 and NHE3 predominantly in the stomach, intestinal tract, and kidneys; NHE5 in the brain; and NHE6–9 ubiquitously in intracellular organelles; whereas NHE10 is specifically expressed in sperm (3, 31). zNHE1 and rainbow trout β-NHE are similar homologs according to the phylogenetic tree analysis, and they were expressed in erythrocytes, which differs from mammalian NHE1. This result seems to be associated with the erythrocyte-specific expression of zebrafish CA2b (Lin TY, Liao BK, Horng JL, Yan JJ, Hsiao CD, Huang PP; unpublished data) and AE1 (33), implying their roles in acid-base regulation of erythrocytes. Similar to mammalian NHE3 (31), zNHE3a and zNHE3b were primarily expressed in kidneys, but only zNHE3b was expressed in gills. The zNHE2 and zNHE3b are the main isoforms expressed in gills and kidneys, and zNHE2 was also weakly expressed in the intestine and kidneys. Different from these, mammalian NHE2 and NHE3 are abundantly expressed in the intestine and kidneys (31). Taken together, zNHE3 is similar to its mammalian counterpart and may play a crucial role in Na+ reabsorption and acid-base regulation in the kidneys; however, differences in functions between zNHE3a and zNHE3b are still unclear. zNHE5 exhibited much greater expression in gills than in the brain, indicating that it may play a more important role in the gills than in the central nervous system in zebrafish. According to the phylogenetic tree analysis, zNHE6–8 were clustered into the same group as were the mammalian NHE6–8, which are intracellular organelle types, but only zNHE8 had the same ubiquitous expression pattern as does the mammalian NHE8 (31). Indeed, further studies are needed to understand the specific roles of NHE6 and NHE7 in various tissues of zebrafish.

Previous studies used heterologous antibodies, i.e., anti-mammalian NHE2 and NHE3 antibodies, to colocalize NHE2 and NHE3 with NKA (a marker for MR cells) in gills of several species (12, 42). The results of NHE2 and NHE3 expressions in gill MR cells should be reconsidered before the antibody specificity is carefully confirmed and/or in situ hybridization with isoform-specific probes are conducted. In the present study, seven isoforms of zNHE were expressed in zebrafish gill tissues, which contain MR cells, ionocytes, epithelial cells, blood cells, nerve cells, and muscle cells. However, isoform-specific in situ hybridization and immunocytochemistry provided convincing molecular evidence that only zNHE3b is expressed in a specific type of ionocytes, HR cells, in zebrafish gills. The antibody used was raised against the dace NHE3 (17), whose epitope shares 68% identity and 75% similarity to the COOH-terminal 19 amino acids of zNHE3b but only 25% identity to that of zNHE3a. Furthermore, zebrafish embryo in situ hybridization revealed that znhe3a and znhe3b were expressed in the middle and posterior pronephric duct, respectively, and accordingly, the dace NHE3 antibody showed immunoreactivity only in the posterior pronephric duct (data not shown). The consistency of zNHE3b in situ hybridization and immunocytochemistry in the posterior pronephric duct of zebrafish embryo further demonstrated the specificity of the dace NHE3 antibody to zHNE3b.

The isoform-specific in situ hybridization and immunocytochemical data provide convincing molecular evidence of the ion transporters and enzymes involved in Na+ uptake and acid-base regulation in zebrafish gill ionocytes. In the present study, zNHE3b was expressed in the apical membrane of HR cells, which differ from NaR cells. mRNAs of two ca isoforms, ca4-like (accession no. EF591981) and ca2 (accession no. NM_199215), were colocalized in the same HR cells in zebrafish (14, 20, 26). In Japanese dace and trout, a homolog of the mammalian Na+/HCO3– cotransporter (pNBC1/NBCe1-B) was cloned and localized immunocytochemically in the basolateral membrane of gill MR cells (17, 32). Preliminary experiments also indicated a homolog of the mammalian NBC1 (accession no. EF634453; Lee YC, Yau JJ, Huang PP; unpublished data) was expressed in zebrafish gill cells. Taking all these together, HR cells in zebrafish appear to be similar to ionocytes of the mammalian renal proximal tubules (38) in the expression patterns of relevant transporters and enzymes, implying similar functions in Na+ uptake and acid-base regulation.

Na+ uptake via the apical NHE in fish gill cells has been questioned for a long time (15, 20, 35). In mammals, genetic knockout of NHE2 had no effect on renal function, whereas complete or kidney-specific knockout of NHE3 resulted in a reduction of proximal tubular Na+ and water loss, demonstrating the specific roles of NHE in Na+ uptake and bicarbonate reabsorption (36, 43). In zebrafish, a similar role of NHE in Na+ uptake was also proposed on the basis of some physiological and pharmacological experiments. Boisen et al. (4) found that amiloride at 10–4 M (a dose that inhibits NHE) inhibited ~40% of the 22Na+ influx in zebrafish (4). Esaki et al. (14) also demonstrated that amiloride at 10–4 M and EIPA, a specific NHE inhibitor, at 10–5 M blocked Na+ accumulation (estimated from the sodium-green fluorescent reagent) and 22Na+ influx in skin HR cells of zebrafish embryos. The present study provides molecular evidence of zNHE3b, supporting those physiological and pharmacological data. On the other hand, a noninvasive ion-selective electrode was used to demonstrate the in vivo function of bafilomycin-sensitive acid secretion from the apical membrane of HR cells in zebrafish embryonic skin (26). Subsequently, knockdown of atp6v1a (HA A-subunit) translation was found to impair both acid secretion and Na+ uptake in zebrafish embryo, providing molecular evidence for the role of HA in the Na+ uptake mechanism (19). Based on these studies, the two pathways, HA/ENaC and NHE, exist and may operate at the same time for Na+ uptake/acid-base regulation in zebrafish HR cells if a homolog or equivalent of mammalian ENaC exists in ionocytes of fish gills.

These two pathways seem to be operated differentially depending on the environmental situation to which fish acclimate. In a recent study by Boisen et al. (4), responses of Na+ uptake to several inhibitors, bafilomycin (for HA), ethoxzolamide (for CA), amiloride (for ENaC and NHE), and EIPA (for NHE), varied in fish acclimated to soft water (35 µM [Na+], 43 µM [Cl–], and 4.4 µM [Ca2+]; pH 6.0) and hard water (1,480 µM [Na+], 1,265 µM [Cl–], and 3,246 µM [Ca2+]; pH 8.2). Craig et al. (12) also found that the mRNA expression of NHE2 in zebrafish gills increased fivefold after expose to soft water for 6 days. NHE2 is not specifically expressed in gill HR cells, and thus its role in the function of HR cells is unclear. Although the ion levels and pH in the media changed in parallel, Boisen et al. (4) pointed out the possibility that two separate mechanisms for Na+ uptake are operating in zebrafish depending on ambient conditions. The present study provides molecular physiological evidence to support this notion. The ion levels ([Na+] and [Cl–]) and pH of the experimental media were independently manipulated to distinguish their specific effects on the expressions (and thus the functions) of znhe3b and atp6v0c. Our molecular physiological experiments found that a low-Na+ environment upregulated znhe3b and downregulated atp6v0c, whereas an acidic environment showed reverse effects, i.e., downregulation of znhe3b and upregulation of atp6v0c.

Taking all these findings together, a model for Na+ uptake and acid-base regulation in zebrafish gill ionocytes is proposed (Fig. 8). Partitioning of zNHE3b and HA in the Na+ uptake/acid-base regulation mechanisms depends on the environmental situations; in low-Na+ environments, apical HA is downregulated to maintain an intracellular H+ gradient to facilitate Na+ uptake via apical zNHE3b, which is the dominant player, and thus its function is enhanced. In acidic environments, however, HA, the dominant player, is upregulated to enhance H+ secretion to maintain the internal acid-base balance, and zNHE3b is greatly downregulated because the ambient high H+ does not favor its operation.


Figure 8
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  		Fig. 8. A proposed model for the differential regulation of NHE3b and HA in zebrafish gill H+-ATPase-rich (HR) cells during acclimation to an acidic or low-Na+ environment. The model was mainly modified from Hwang and Lee (20). See text for details. Sizes of HA and NHE reflect their relative levels of expressions. Shading indicates pathways proposed from previous studies (17, 20, 32). CA2 and CA4, carbonic anhydrase isoforms; NBC, Na+/HCO3– cotransporter; NHE, Na+/H+ exchanger 3b.

 
In summary, the present study used functional genomic approaches to identify only one member, zNHE3b, from the zNHE family, which is specifically expressed in gill HR cells and is functionally regulated in response to environmental acidic or low-Na+ challenges. This molecular and cellular physiological evidence not only provides new insights into the mechanisms of Na+ uptake/acid-base regulation in gill ionocytes but also demonstrates that zebrafish are an excellent in vivo model for research on the transport physiology of human proximal tubule based on similarities in the expressions of relevant transporters and enzymes.


   ACKNOWLEDGMENTS
 
We thank Y. C. Tung and J. I. Wang for assistance during the experiments and the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, for assistance in sequencing and confocal microscopy.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. P. Hwang, Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China (e-mail: pphwang{at}gate.sinica.edu.tw)

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. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Amores A, Force A, Yan YL, Joly L, Amemiya C, Fritz A, Ho RK, Langeland J, Prince V, Wang YL, Westerfield M, Ekker M, Postlethwait JH. Zebrafish hox clusters and vertebrate genome evolution. Science 282: 1711–1714, 1998.[Abstract/Free Full Text]

2. Avella M, Bornancin M. A new analysis of ammonia and sodium-transport through the gills of the fresh-water rainbow-trout (Salmo gairdneri). J Exp Biol 142: 155–175, 1989.[Abstract/Free Full Text]

3. Bobulescu IA, Di Sole F, Moe OW. Na+/H+ exchangers: physiology and link to hypertension and organ ischemia. Curr Opin Nephrol Hypertens 14: 485–494, 2005.[Web of Science][Medline]

4. Boisen AM, Amstrup J, Novak I, Grosell M. Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio). Biochim Biophys Acta 1618: 207–218, 2003.[Medline]

5. Canfield VA, Loppin B, Thisse B, Thisse C, Postlethwait JH, Mohideen MA, Rajarao SJ, Levenson R. Na,K-ATPase alpha and beta subunit genes exhibit unique expression patterns during zebrafish embryogenesis. Mech Dev 116: 51–59, 2002.[CrossRef][Web of Science][Medline]

6. Capasso G, Cantone A, Evangelista C, Zacchia M, Trepiccione F, Acone D, Rizzo M. Channels, carriers, and pumps in the pathogenesis of sodium-sensitive hypertension. Semin Nephrol 25: 419–424, 2005.[CrossRef][Web of Science][Medline]

7. Chan YL, Giebisch G. Relationship between sodium and bicarbonate transport in the rat proximal convoluted tubule. Am J Physiol Renal Fluid Electrolyte Physiol 240: F222–F230, 1981.[Abstract/Free Full Text]

8. Choi JY, Shah M, Lee MG, Schultheis PJ, Shull GE, Muallem S, Baum M. Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule. J Clin Invest 105: 1141–1146, 2000.[Web of Science][Medline]

9. Claiborne JB, Edwards SL, Morrison-Shetlar AI. Acid-base regulation in fishes: cellular and molecular mechanisms. J Exp Zool 293: 302–319, 2002.[CrossRef][Web of Science][Medline]

10. Counillon L, Franchi A, Pouyssegur J. A point mutation of the Na+/H+ exchanger gene (NHE1) and amplification of the mutated allele confer amiloride resistance upon chronic acidosis. Proc Natl Acad Sci USA 90: 4508–4512, 1993.[Abstract/Free Full Text]

11. Counillon L, Noel J, Reithmeier RA, Pouyssegur J. Random mutagenesis reveals a novel site involved in inhibitor interaction within the fourth transmembrane segment of the Na+/H+ exchanger-1. Biochemistry 36: 2951–2959, 1997.[CrossRef][Medline]

12. Craig PM, Wood CM, McClelland GB. Gill membrane remodeling with soft-water acclimation in zebrafish (Danio rerio). Physiol Genomics 30: 53–60, 2007.[Abstract/Free Full Text]

13. Edwards SL, Tse CM, Toop T. Immunolocalisation of NHE3-like immunoreactivity in the gills of the rainbow trout (Oncorhynchus mykiss) and the blue-throated wrasse (Pseudolabrus tetrious). J Anat 195: 465–469, 1999.[CrossRef][Web of Science][Medline]

14. Esaki M, Hoshijima K, Kobayashi S, Fukuda H, Kawakami K, Hirose S. Visualization in zebrafish larvae of Na+ uptake in mitochondria-rich cells whose differentiation is dependent on foxi3a. Am J Physiol Regul Integr Comp Physiol 292: R470–R480, 2007.[Abstract/Free Full Text]

15. Evans DH, Piermarini PM, Choe KP. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85: 97–177, 2005.[Abstract/Free Full Text]

16. Higgins DG, Sharp PM. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73: 237–244, 1988.[CrossRef][Web of Science][Medline]

17. Hirata T, Kaneko T, Ono T, Nakazato T, Furukawa N, Hasegawa S, Wakabayashi S, Shigekawa M, Chang MH, Romero MF, Hirose S. Mechanism of acid adaptation of a fish living in a pH 3.5 lake. Am J Physiol Regul Integr Comp Physiol 284: R1199–R1212, 2003.[Abstract/Free Full Text]

18. Hirose S, Kaneko T, Naito N, Takei Y. Molecular biology of major components of chloride cells. Comp Biochem Physiol B Biochem Mol Biol 136: 593–620, 2003.[CrossRef][Medline]

19. Horng JL, Lin LY, Huang CJ, Katoh F, Kaneko T, Hwang PP. Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio). Am J Physiol Regul Integr Comp Physiol 292: R2068–R2076, 2007.[Abstract/Free Full Text]

20. Hwang PP, Lee TH. New insights into fish ion regulation and mitochondrion-rich cells. Comp Biochem Physiol A Mol Integr Physiol 148: 479–497, 2007.[CrossRef][Medline]

21. Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, Bouneau L, Fischer C, Ozouf-Costaz C, Bernot A, Nicaud S, Jaffe D, Fisher S, Lutfalla G, Dossat C, Segurens B, Dasilva C, Salanoubat M, Levy M, Boudet N, Castellano S, Anthouard V, Jubin C, Castelli V, Katinka M, Vacherie B, Biemont C, Skalli Z, Cattolico L, Poulain J, De Berardinis V, Cruaud C, Duprat S, Brottier P, Coutanceau JP, Gouzy J, Parra G, Lardier G, Chapple C, McKernan KJ, McEwan P, Bosak S, Kellis M, Volff JN, Guigo R, Zody MC, Mesirov J, Lindblad-Toh K, Birren B, Nusbaum C, Kahn D, Robinson-Rechavi M, Laudet V, Schachter V, Quetier F, Saurin W, Scarpelli C, Wincker P, Lander ES, Weissenbach J, Roest Crollius H. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431: 946–957, 2004.[CrossRef][Medline]

22. Katoh F, Hyodo S, Kaneko T. Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment. J Exp Biol 206: 793–803, 2003.[Abstract/Free Full Text]

23. Khadilkar A, Iannuzzi P, Orlowski J. Identification of sites in the second exomembrane loop and ninth transmembrane helix of the mammalian Na+/H+ exchanger important for drug recognition and cation translocation. J Biol Chem 276: 43792–43800, 2001.[Abstract/Free Full Text]

24. Kirschner LB. Sodium-chloride absorption across the body-surface - frog skins and other epithelia. Am J Physiol Regul Integr Comp Physiol 244: R429–R443, 1983.[Abstract/Free Full Text]

25. Lin H, Randall DJ. Evidence for the presence of an electrogenic proton pump on the trout gill epithelium. J Exp Biol 161: 119–134, 1991.[Abstract/Free Full Text]

26. Lin LY, Horng JL, Kunkel JG, Hwang PP. Proton pump-rich cell secretes acid in skin of zebrafish larvae. Am J Physiol Cell Physiol 290: C371–C378, 2006.[Abstract/Free Full Text]

27. Lin YM, Chen CN, Yoshinaga T, Tsai SC, Shen ID, Lee TH. Short-term effects of hyposmotic shock on Na+/K+-ATPase expression in gills of the euryhaline milkfish, Chanos chanos. Comp Biochem Physiol A Mol Integr Physiol 143: 406–415, 2006.[CrossRef][Medline]

28. Marshall WS. Na+, Cl–, Ca2+ and Zn2+ transport by fish gills: retrospective review and prospective synthesis. J Exp Zool 293: 264–283, 2002.[CrossRef][Web of Science][Medline]

29. Meng H, Pierce GN. Involvement of sodium in the protective effect of 5-(N,N-dimethyl)-amiloride on ischemia-reperfusion injury in isolated rat ventricular wall. J Pharmacol Exp Ther 256: 1094–1100, 1991.[Abstract/Free Full Text]

30. Nelson ID, Potts WTW, Huddart H. The use of amiloride to uncouple branchial sodium and proton fluxes in the brown trout, Salmo trutta. J Comp Physiol [B] 167: 123–128, 1997.[CrossRef]

31. Orlowski J, Grinstein S. Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflügers Arch 447: 549–565, 2004.[CrossRef][Web of Science][Medline]

32. Parks SK, Tresguerres M, Goss GG. Interactions between Na+ channels and Na+-HCO3– cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake. Am J Physiol Cell Physiol 292: C935–C944, 2007.[Abstract/Free Full Text]

33. Paw BH, Davidson AJ, Zhou Y, Li R, Pratt SJ, Lee C, Trede NS, Brownlie A, Donovan A, Liao EC, Ziai JM, Drejer AH, Guo W, Kim CH, Gwynn B, Peters LL, Chernova MN, Alper SL, Zapata A, Wickramasinghe SN, Lee MJ, Lux SE, Fritz A, Postlethwait JH, Zon LI. Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency. Nat Genet 34: 59–64, 2003.[CrossRef][Web of Science][Medline]

34. Perry SF, Randall DJ. Effects of amiloride and SITS on branchial ion fluxes in rainbow trout, Salmo gairdneri. J Exp Zool 215: 225–228, 1981.[CrossRef][Web of Science][Medline]

35. Perry SF, Shahsavarani A, Georgalis T, Bayaa M, Furimsky M, Thomas SL. Channels, pumps, and exchangers in the gill and kidney of freshwater fishes: their role in ionic and acid-base regulation. J Exp Zoolog A Comp Exp Biol 300: 53–62, 2003.[Medline]

36. Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger. Nat Genet 19: 282–285, 1998.[CrossRef][Web of Science][Medline]

37. Touret N, Poujeol P, Counillon L. Second-site revertants of a low-sodium-affinity mutant of the Na+/H+ exchanger reveal the participation of TM4 into a highly constrained sodium-binding site. Biochemistry 40: 5095–5101, 2001.[CrossRef][Medline]

38. Wagner CA, Finberg KE, Breton S, Marshansky V, Brown D, Geibel JP. Renal vacuolar-ATPase. Physiol Rev 84: 1263–1314, 2004.[Abstract/Free Full Text]

39. Wakabayashi S, Hisamitsu T, Pang T, Shigekawa M. Mutations of Arg440 and Gly455/Gly456 oppositely change pH sensing of Na+/H+ exchanger 1. J Biol Chem 278: 11828–11835, 2003.[Abstract/Free Full Text]

40. Wang D, King SM, Quill TA, Doolittle LK, Garbers DL. A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility. Nat Cell Biol 5: 1117–1122, 2003.[CrossRef][Web of Science][Medline]

41. Wang T, Yang CL, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, Aronson PS. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. Am J Physiol Renal Physiol 277: F298–F302, 1999.[Abstract/Free Full Text]

42. Wilson JM, Laurent P, Tufts BL, Benos DJ, Donowitz M, Vogl AW, Randall DJ. NaCl uptake by the branchial epithelium in freshwater teleost fish: an immunological approach to ion-transport protein localization. J Exp Biol 203: 2279–2296, 2000.[Abstract]

43. Woo AL, Noonan WT, Schultheis PJ, Neumann JC, Manning PA, Lorenz JN, Shull GE. Renal function in NHE3-deficient mice with transgenic rescue of small intestinal absorptive defect. Am J Physiol Renal Physiol 284: F1190–F1198, 2003.[Abstract/Free Full Text]

44. Wright PA, Wood CM. An analysis of branchial ammonia excretion in the fresh-water rainbow-trout: effects of environmental pH change and sodium uptake blockade. J Exp Biol 114: 329–353, 1985.[Abstract/Free Full Text]




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T.-Y. Lin, B.-K. Liao, J.-L. Horng, J.-J. Yan, C.-D. Hsiao, and P.-P. Hwang
Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish H+-ATPase-rich cells
Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1250 - C1260.
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