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Am J Physiol Regul Integr Comp Physiol 294: R1402-R1412, 2008. First published January 23, 2008; doi:10.1152/ajpregu.00759.2007
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WATER AND ELECTROLYTE HOMEOSTASIS

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Yukihiro Kurita,1 Tsutomu Nakada,1 Akira Kato,1 Hiroyuki Doi,2 Abinash C. Mistry,1 Min-Hwang Chang,3 Michael F. Romero,3 and Shigehisa Hirose1

1Department of Biological Sciences, Tokyo Institute of Technology, Yokohama; 2Shimonoseki Marine Science Museum "Kaikyokan," Shimonoseki Academy of Marine Science, Shimonoseki, Japan; and 3Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 18 October 2007 ; accepted in final form 19 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3– to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3– secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3– transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3– cotransporter) and Slc26a6A and Slc26a6B (putative Cl–/HCO3– exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3–-transporting activity as electrogenic Cl–/nHCO3– exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3– cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3– secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3– transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3– uptake (NBCe1) and apical HCO3– secretion (Slc26a6A and Slc26a6B).

osmoregulation; NBC; Slc26; intestine; calcium carbonate

THE BODY FLUID OF MOST MARINE teleosts (~300 mosmol/l) is hyposmotic to seawater (SW, ~1,000 mosmol/l). To avoid dehydration, therefore, marine fishes drink the ambient SW, absorb water, and eliminate salt through the following processes (29). The ingested SW is first partially desalted in the esophagus by passive diffusion before entering the stomach, and desalting continues in the intestine by active transport of monovalent ions from the lumen into the blood. The absorbed monovalent ions (mainly Na+ and Cl–) are then excreted through the gill. The desalting process reduces the osmolarity of the ingested SW and allows passive uptake of water across the intestinal epithelium. Divalent cations (Ca2+ and Mg2+) are also present in SW and concentrated to extremely high levels as water absorption proceeds in the intestine. Notably, the divalent cations left behind are precipitated as carbonate deposits inside the intestinal lumen and excreted with rectal fluid (47). The water-insoluble carbonate precipitates are mainly composed of CaCO3 and MgCO3 and seen in virtually all marine teleosts (39, 47–49). Wilson et al. (51) proposed that this precipitation facilitates water absorption by effectively removing osmolytes from the intestinal lumen that would otherwise accumulate and retard water absorption. The carbonate deposit formation thus appears to play an important role in osmoregulation in marine fish. Evidence for this role includes 1) the presence of carbonate deposits in marine teleosts and its absence in their fresh-water (FW) counterparts (47), 2) the fact that the ingested SW becomes alkaline (pH 8.4–9.0) and rich in HCO3– as it transits the intestine, a condition that is suitable for carbonate deposit formation and occurs even when marine teleosts are deprived of food (39, 45, 46), 3) the presence of a Cl–/HCO3– exchanger activity and immunoreactivity on the apical membrane of intestinal epithelial cells of marine fish, which secrete HCO3– concomitantly with Cl– absorption (4, 15, 51), and 4) the demonstration that the rate of HCO3– secretion is stimulated by increased amounts of Ca2+ entering the intestine (51). Nonteleosts (Acipenser baerii, Chiloscyllium plagiosum, and Lampetra fluviatilis) also show intestinal HCO3– secretion under hyperosmotic conditions, suggesting the evolutionary conservation of the system (44). However, the molecular identity of the HCO3– transporter(s) involved remains undetermined.

We recently focused on a euryhaline fugu species, mefugu (Takifugu obscurus) (1), which lives on the seacoasts and in river mouth areas of China and Korea and migrates to brackish water and FW for spawning (21). Mefugu can live in FW and SW (20) and is therefore classified as a secondary FW fish. Mefugu may be an ideal fish species for studying FW and SW adaptation of fish, since the complete genome sequence of the tiger puffer Takifugu rubripes, a close relative of mefugu, is available. In the present study, we attempted to identify the HCO3– transporter(s) responsible for carbonate deposit formation by assuming that they are induced in the intestinal epithelial cells when mefugu is transferred from FW to SW. First, we isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO3– from the mefugu T. obscurus on the basis of the DNA sequence information on the T. rubripes genome. Then we performed Northern blot analyses to select clones highly expressed in the intestine of SW mefugu. Finally, we determined the cellular locations of their protein products by immunohistochemistry. On the basis of these observations, we proposed a model for HCO3– excretion across the intestinal epithelium of marine teleost fish that includes a basolateral Na+-nHCO3– cotransporter (NBCe1 or Slc4A4) and apical Cl–/HCO3– exchangers (Slc26a6A and Slc26a6B). CaCO3 and MgCO3 deposit formation is central to the maintenance of body fluid homeostasis in marine fish and a substantial source of marine CaCO3 sediments (47).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Mefugu T. obscurus (245–360 g) were purchased from a local dealer and reared in 150-liter brackish water (3–14% diluted SW) tanks. Mefugu were then transferred to 150-liter FW tanks for 8–9 days and used as FW mefugu. Mefugu in the FW tanks were then transferred to 150-liter SW tanks and acclimated for 1, 3, and 8 days (SW mefugu). Water temperature was maintained at 18–22°C. All fugu were anesthetized by immersion in 0.1% ethyl m-aminobenzoate (MS-222 or tricaine) before they were killed by decapitation. The tissues required for RNA extraction were dissected out, snap frozen in liquid nitrogen, and stored at –80°C. Artificial SW (Rohtomarine) was obtained from Rei-Sea (Tokyo, Japan). The animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of the Tokyo Institute of Technology and conformed to the "Guiding Principles in the Care and Use of Laboratory Animals" of the American Physiological Society (3).

RNA isolation. Total RNA was isolated from the intestine by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen (Nippon Gene, Tokyo, Japan). Tissues were homogenized in Isogen (1 g of tissue per 10 ml of Isogen) using a Polytron tissue homogenizer and subjected to chloroform extraction and isopropanol precipitation. Precipitated RNA was washed with 75% (vol/vol) ethanol and dissolved in diethyl pyrocarbonate-treated water, and its concentration was measured spectrophotometrically at 260 nm.

Molecular cloning. cDNA was reverse transcribed using random hexamers and the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Fragments of mefugu NBCe1, Slc26a6A, and Slc26a6B were isolated by RT-PCR from fugu intestine RNA with primers (see Table 1 in supplemental data for this article available online at the American Journal of Physiology-Regulatory, Integrative, and Comparative Physiology website) that were designed on the basis of the fugu genomic database (http://genome.jgi-psf.org/fugu6/fugu6.home.html). The PCR products were subcloned into pBluescript II (SK–) (Stratagene, La Jolla, CA) and sequenced. These clones were used as probes for Northern blot analysis. Full-length cDNAs were obtained by rapid amplification of cDNA ends (RACE) using the First Choice RLM-RACE kit (Ambion, Austin, TX) according to the manufacturer's instructions (see list of all primers in Table 1 in the online version of this article).


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  		Table 1. Tiger puffer (Takifugu rubripes) cDNA clones that are homologous to SLC4 and SLC26 family members

 
Northern blot analysis. Total RNA (20 µg/lane) from a pool of various tissues of FW and SW mefugu was separated by electrophoresis on formaldehyde-agarose (1%) denaturing gels in 10x MOPS running buffer [20 mM MOPS (pH 7.0), 8 mM acetate, and 1 mM EDTA] and then transferred onto Hybond-N+ nylon membranes (GE Healthcare, Piscataway, NJ) by capillary blotting. The membranes were baked for 2 h at 80°C and prehybridized for 2 h at 65°C in PerfectHyb hybridization solution (Toyobo, Osaka, Japan). The probes were labeled with [{alpha}-32P]dCTP (3,000 Ci/mmol) with a Ready-To-Go DNA labeling kit (GE Healthcare), and the unincorporated nucleotides were removed by passage through a Sephadex G-50 column (GE Healthcare). The membranes were then hybridized separately with each 32P-labeled probe in the same buffer at 68°C for 16 h. The blots were subsequently washed under increasingly stringent conditions, with the final wash in 1x saline-sodium citrate and 0.1% SDS for 30 min at 60°C. Membranes were exposed to an imaging plate (Fuji Film, Tokyo, Japan) in a cassette overnight. The results were analyzed using a Bio-Image analyzer (model BAS2000, Fuji Film). A probe for mefugu β-actin was used as a control to verify loading and RNA integrity.

Real-time PCR. Expression of mfNBCe1, mfSlc26a6A, and mfSlc26a6B was quantified by real-time PCR. Total RNAs were extracted from segment a of the intestine (Fig. 1B), which was individually excised from mefugu acclimated to SW and FW (n = 5 for each group), treated with DNase (RNase-free DNase set, Qiagen, Valencia, CA), and then purified on spin columns (RNeasy MinElute Cleanup Kit, Qiagen). cDNA was reverse transcribed using oligo(dT) primer and the SuperScript III First-Strand Synthesis System (Invitrogen) according to the manufacturer's instruction. Multiplex real-time PCR was performed for quantitation of mfNBCe1, mfSlc26a6A, and mfSlc26a6B mRNA expression, with amplification of GAPDH as an endogenous control; TaqMan analysis and subsequent calculations were performed with a sequence detection system (ABI Prism 7900HT, Applied Biosystems, Foster City, CA), which detects the increase in fluorescent signal released from an internal fluorogenic probe as the PCR proceeds. For each sample, 1 µg of total RNA was subjected to real-time PCR according to the protocol provided by the manufacturer of the TaqMan One-Step PCR Master Mix Reagents Kit (SYBR Green I, Takara Bio, Otsu, Japan). A PCR was also performed on total RNA that had not been reverse transcribed to control for the absence of genomic DNA in the RNA preparation (see Table 1 in the online version of this article for sequences of the forward and reverse primers). mfNBCe1, mfSlc26a6A, and mfSlc26a6B mRNA concentrations were normalized to GAPDH levels. Experiments were performed in triplicate for each standard.


Figure 1
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  		Fig. 1. Divalent cation precipitates formed in intestine of seawater (SW) mefugu Takifugu obscurus. A: intestinal segment excised and cut longitudinally for visualization of divalent cation precipitates. B: mefugu intestine divided into 4 regions from anterior to posterior (sections a–d) and subjected to Northern analysis (Fig. 2A, left). Takifugu and mefugu are agastric fish species.

 
Expression of mfNBCe1, mfSlc26a6A, and mfSlc26a6B in Xenopus oocytes and electrophysiology. The entire coding regions of mfNBCe1, mfSlc26a6A, and mfSlc26a6B cDNAs were inserted into the pGEMHE Xenopus laevis expression vector (26). The Slc26a6A and Slc26a6B plasmids were linearized with Not I and Nhe I, respectively, and cRNA was transcribed in vitro using T7 RNA polymerase and mMESSAGE mMACHINE kits (Ambion). X. laevis oocytes were dissociated with collagenase as previously described (36) and injected with 25 nl of water or a solution containing cRNA at 1 µg/µl (25 ng/oocyte) using a Nanoject-II injector (Drummond Scientific, Broomall, PA). Oocytes were incubated at 16°C in OR3 medium (36) and studied 3–6 days after injection.

For measurement of intracellular pH (pHi), an H+-selective microelectrode was prepared with an H+ ionophore I-mixture B ion-selective resin (Fluka Chemical) and used as previously described (36). pHi was measured as the difference between the pH electrode and a KCl voltage electrode impaled into the oocyte, and membrane potential (Vm) was measured as the difference between the KCl microelectrode and an extracellular calomel (36). pH electrodes were calibrated using pH 6.0 and 8.0 buffer (Fisher Scientific), and point calibration was carried out in ND96 solution (pH 7.50) as described previously (36); slopes were at least –54 mV/pH unit. ND96 solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES (pH 7.5). For CO2/HCO3–-equilibrated solutions, 33 mM NaHCO3 was replaced with 33 mM NaCl, and the solutions were bubbled with 5% CO2-95% O2 during the experiments. In 0-Cl– solutions, Cl– was replaced by gluconate, and choline replaced Na+ in 0-Na+ solutions.

Antibody production and specificity. For preparation of recombinant proteins for use as antigens, cDNA fragments encoding a part of mfNBCe1 [amino acid (aa) residues 985-1075], mfSlc26a6A (aa residues 651-771), and mfSlc26a6B (aa residues 601-706) were cloned into the bacteria expression vector pHAT10 (Clontech, Palo Alto, CA). These plasmids were introduced into the Escherichia coli BL21 strain (Codon Plus). The cells were grown in Luria-Bertani broth containing 100 µg/ml ampicillin to an absorbance at 600 nm of 0.55–0.60 at 37°C, and protein expression was induced by addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM for 6 h. The cells were harvested by centrifugation at 2,000 g for 5 min, and the cell pellets were washed once with ice-cold 10 mM PBS (pH 7.4) and resuspended in wash buffer [50 mM Na2PO4 and 300 mM NaCl (pH 7.0)]. After disruption of the cells by a freeze-thaw cycle and sonication, the lysates were centrifuged at 12,000 g for 20 min, with the insoluble fractions recovered as a pellet. In a pilot experiment, all the desired recombinant proteins were found in the insoluble fractions. The insoluble fractions were solubilized, and recombinant proteins were purified with a metal affinity resin (BD TALON, Clontech) according to the manufacturer's instructions and dialyzed against saline [0.9% (wt/vol) NaCl] at 4°C. Polyclonal antibodies were prepared in Japanese white rabbits by intramuscular injection of ~200 µg of purified recombinant protein, emulsified with the adjuvant TiterMax Gold (CytRx, Norcross, GA; 1:1), at 1-mo intervals, with the rabbits bled 7 days after the third immunization.

Antibody specificity was established by staining of COS-7 cells exogenously expressing the corresponding antigen proteins (see Fig. S1 in the online version of this article). The cDNA fragments corresponding to the open reading frames of mfNBCe1, mfSlc26a6A, and mfSlc26a6B were subcloned into the p3xFLAG-CMV-10 (Sigma-Aldrich, Tokyo, Japan) vector. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) containing 10% FBS (Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were transfected with the mfNBCe1-FLAG, mfSlc26a6A-FLAG, mfSlc26a6B-FLAG, or mock plasmids with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The cells were fixed with 3.7% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 1% FBS in Hanks' balanced salt solution for 30 min at room temperature. They were incubated with primary antibodies (1:1,000 dilution, or 1.2 µg/ml for anti-FLAG) for 1 h at room temperature and then with appropriate secondary antibodies conjugated with Alexa 350 or Alexa 546 (1:2,000 dilution; Invitrogen) for 30 min.

In situ hybridization. Segment a of intestine (Fig. 1B) from SW mefugu was perfused and fixed with 10% buffered neutral formalin (Muto Pure Chemicals, Tokyo, Japan), harvested, embedded in paraffin, and sectioned (4 µm). The following DNA templates were used for preparation of digoxigenin (DIG)-labeled riboprobes: a 1,227-bp fragment of mfNBCe1 cDNA [nucleotides (nt) 1805–3031], a 548-bp fragment of mfSlc26a6A cDNA (nt 1997–2544), and a 397-bp fragment of mfSlc26a6B cDNA (nt 1834–2230). A DIG RNA labeling mix (Roche Diagnostics, Mannheim, Germany) was used for synthesis of DIG-labeled sense and antisense probes. Alkaline phosphatase-conjugated anti-DIG antibodies and nitro blue tetrazolium-bromochloroindolyl phosphate substrates were used to visualize the signal; then the sample was counterstained with Kernechtrot (Muto Pure Chemicals).

Immunohistochemistry. Segment a of intestine (Fig. 1B) from SW mefugu was fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% (wt/vol) paraformaldehyde for 1 h at 4°C. After incubation in 0.1 M phosphate buffer (pH 7.4) containing 20% (wt/vol) sucrose for 16 h at 4°C, specimens were frozen in Tissue Tek OCT compound on a cryostat holder. Sections (6 µm) were prepared on a –20°C cryostat and mounted on (3-aminopropyl)triethoxysilane-coated glass slides and air-dried for 1 h. The sections were washed with PBS and then incubated for 2 h at room temperature with 2.5% (vol/vol) normal goat serum and overnight at 4°C with antiserum and preimmune serum [1:10,000 dilution for the ABC method (avidin-biotin-peroxidase complex) or 1:1,000 dilution for the immunofluorescence method]. The ABC method was performed as follows. The sections were incubated with antiserum and preimmune serum and then washed with PBS and treated with methanol containing 0.3% (vol/vol) H2O2 for 20 min at room temperature. After they were washed with PBS, the specimens were treated with biotinylated secondary antibody (1:2,000 dilution) and avidin-peroxidase conjugate using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Finally, bound antibodies were visualized with 3,3'-diaminobenzidine tetrahydrochloride in 50 mM Tris·HCl (pH 7.4) and counterstained with hematoxylin. For immunofluorescence, sections labeled with primary antibodies were washed with PBS and then incubated with a cocktail mix containing Alexa Fluor 594-conjugated anti-rabbit IgG (1:2,000 dilution; Invitrogen) and TO-PRO3 (2 µM; Invitrogen) for 1 h at room temperature. Fluorescence was detected using a confocal microscope (model LSM 5, Carl Zeiss, Oberkochen, Germany). Images were obtained with a high-resolution digital charge-coupled device camera and processed with an LSM5 image browser (Carl Zeiss).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Carbonate deposit formation in SW mefugu intestine. Consistent with the previous observations suggesting that the formation of intestinal carbonate deposits is a general feature of osmoregulation in marine teleost fishes (41), carbonate precipitates were contained in the intestinal lumen of all SW-adapted mefugu examined (Fig. 1A; n = 14). In the case of FW mefugu, this carbonate deposit was completely absent (n = 6). Similar carbonate deposit formation was also observed in the intestine of euryhaline eels (Angulla japonica) when they were transferred from FW to SW (n = 12).

Identification of candidate HCO3– transporters by database mining. The database of the Human Genome Organisation (HUGO) Nomenclature Committee provides a list of 46 solute transporter families and 360 transporter genes (http://www.gene.ucl.ac.uk/nomenclature/ and http://www.bioparadigms.org/slc/menu.asp) (18). Members of the solute carrier families SLC4 (37) and SLC26 (33) have HCO3– transport activities. We therefore searched the T. rubripes genome sequence for homologs of the human SLC4 and SLC26 family members and identified 24 candidate sequences in the fugu genome (Table 1).

Selection of candidate clones by Northern blot analysis and real-time PCR. Partial cDNA clones for the 24 candidate transporters predicted by database mining (14 Slc4 family members and 10 Slc26 family members; Table 1) were obtained by RT-PCR using RNA preparations from gill, kidney, and intestine of SW mefugu, sequenced, and used as probes for Northern blot analysis. Among the candidates, three clones were highly expressed in intestine of SW mefugu: Slc4a4 (NBCe1) and Slc26a6A and Slc26a6B (PAT-1/CFEX; Fig. 2). Notably, mfNBCe1 and mfSlc26a6A mRNA levels were upregulated 2.6- and 3.1-fold, respectively, vs. FW mefugu (Fig. 2C; P < 0.05, n = 3–5), making them good candidates for the transporters involved in intestinal carbonate deposit formation. The intestinal expression of mfSlc26a6B was high in FW and SW mefugu (n = 5), suggesting that mfSlc26a6B may regulate fluid secretion for digestion (45, 46) in FW, as well as intestinal carbonate deposit formation in SW. Interestingly, all three transporters showed high-level expression in the anterior region (segment a) of the intestine in SW mefugu (Fig. 2A), which is consistent with the site of HCO3– secretion in the intestine of other marine teleosts (10, 13).


Figure 2
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  		Fig. 2. Tissue-specific and salinity-dependent expression of mfNBCe1, mfSlc26a6A, and mfSlc26a6B. A: Northern blot analysis of 20 µg of total RNA from tissues of mefugu acclimated to fresh-water (FW, F) or SW (S) for 8 days. B: time course of changes of mRNA levels after transfer of mefugu from FW to SW. Total RNA was isolated from segment a of intestine (see Fig. 1B) and subjected to Northern blot analysis. C: real-time PCR quantification of mfNBCe1, mfSlc26a6A, and mfSlc26a6B expression. Values are means ± SE. *P < 0.05.

 
Cloning and molecular characterization of HCO3– transporters highly expressed in intestine of SW mefugu. To determine the complete nucleotide sequences of mfNBCe1, mfSlc26a6A, and mfSlc26a6B, full-length cDNAs were obtained by 5'- and 3'-RACE using total RNA preparations from SW mefugu intestines. We determined the deduced amino acid sequences (Fig. 3), phylogenetic trees (see Fig. S2 in the online version of this article), and putative membrane topologies (see Fig. S3 in the online version of this article) of the three transporters.


Figure 3
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  		Fig. 3. Phylogenetic trees and structural features of mfNBCe1, mfSlc26a6A, and mfSlc26a6B. A: phylogenetic tree of members of Slc4 family and secondary structure model of mfNBCe1. Phylogenetic tree was constructed using ClustalW computer program. Scale bar represents a genetic distance of 0.1 amino acid substitution per site. mf; mefugu (T. obscurus); h, human; r, rat; m, mouse. Accession numbers are as follows: mfNBCe1 (AB362567), hNBCe1 (NM_001098484), mNBCe1 (NM_018760), rNBCe1 (NM_053424), hNBCe2 (NM_021196), rNBCe2 (NM_212512), hNBCn1 (NM_003615), mNBCn1 (NM_001033270), rNBCn1 (NM_058211), hAE4 (NM_031467), rAE4 (NM_152938), and mAE4 (NM_172830). Putative transmembrane domains of fNBCe1 were predicted by Kyte-Doolittle hydropathy plot. Plot was constructed using the GENETYX computer program. B: phylogenetic tree of members of Slc26 family and secondary structural model of mfSlc26a6A and mfSlc26a6B. z, zebrafish (Danio rerio); e, eel (Anguilla japonica). Accession numbers are as follows: mfSlc26a6A (AB200328), mfSlc26a6B (AB200329), hSlc26a1 (AF297659), eSlc26A1 (AB111927), hSlc26a2 (NM_000112), hSlc26A3 (NM_000111), hSlc26a4 (NM_000441), hSlc26a5 (NM_198999), zSlc26a5 (NM_201473), hSlc26A6 (NM_022911), eSlc26a6 (AB084425), hSlc26a7 (AF331521), hSlc26A8 (AF331522), hSlc26A9 (AF331525), and hSlc26a11 (NM_173626). STAS, sulfate transporter and anti-sigma factor antagonist.

 
Mefugu NBCe1 consists of 1,075 amino acid residues and has 13 predicted transmembrane spans (Fig. 3). In mammals, three types of variants of NBCe1 arise from a single gene by alternative promoter usage and splicing. Two are expressed in epithelial tissues, with the kidney (NBCe1-A or kNBCe1) and pancreatic (NBCe1-B or pNBCe1) isoforms differing only in their length and sequence of the NH2-terminal cytoplasmic tail (41 and 85 aa residues, respectively). A third isoform, with a COOH-terminal variation, exists in the brain (7). The mefugu NBCe1 cloned here is classified as NBCe1-B, with a longer NH2 terminus. The COOH terminus of mefugu NBCe1 contains several conserved regions: the putative binding motifs for carbonic anhydrase (CA) II (aa residues 748-776) (31) and CA-IV (2) and a basolateral targeting motif (QEPML, aa residues 1051-1055) (25). Mefugu Slc26a6A and Slc26a6B belong to the Slc26 family of anion transporters and are composed of 771 and 706 amino acid residues, respectively (Fig. 3). Similar to their mammalian counterparts, they have a sulfate transporter and anti-sigma factor antagonist (STAS) domain and a PDZ-binding motif in their cytoplasmic COOH-terminal tail.

Functional characterization of mfNBCe1. When expressed in Xenopus oocytes, mfNBCe1 mediated an electrogenic, Na+- and HCO3–-dependent pHi change. Addition of CO2/HCO3– elicited a decline of pHi in water-injected controls and mfNBCe1-expressing oocytes (due to CO2 entry into the cell by diffusion; Fig. 4, A and B). In mfNBCe1 oocytes (Fig. 4B), addition of CO2/HCO3– also elicited a hyperpolarization (Na+-nHCO3– into the oocyte, –18.3 ± 2.6 mV, n = 4). Removal of extracellular Na+ depolarized the oocyte slightly (14.6 ± 4.4 mV, n = 4) and decreased pHi (Na+-nHCO3– exit from the oocyte, –5.1 ± 1.8 x 10–4 pH units/s, n = 4). Readdition of Na+ elicited a marked and instantaneous hyperpolarization (due to mfNBCe1-dependent HCO3– transport into the cell, –36.6 ± 8.0 mV, n = 4). These responses were not observed in water-injected oocytes (Fig. 4A). This pattern of response to removal and readdition of Na+ is similar to that of mammalian NBCe1, but the degree of depolarization and hyperpolarization is quite different.


Figure 4
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  		Fig. 4. HCO3– transport mediated by mfNBCe1, mfSlc26a6A, and mfSlc26a6B. Representative traces of intracellular pH (pHi) and membrane potential (Vm) of oocytes injected with water (control, A), mfNBCe1 cRNA (B), mfSlc26a6A cRNA (C), and mfSlc26a6B cRNA (D) are shown. In the continuous presence of 5% CO2/33 mM HCO3– and HCO3–, transport activities were monitored as change in pHi and Vm when extracellular Na+ and Cl– were removed and readded. Solution changes for ND96 with CO2/HCO3–, 0-Cl– ND96 solution with CO2/HCO3–, and 0-Na+ solution ND96 with CO2/HCO3– are indicated by white, gray, and dashed bars, respectively. Numbers adjacent to traces indicate pH slope (10–5 pH units/s).

 
Functional characterization of mfSlc26a6A and mfSlc26a6B. Oocytes were injected with mfSlc26a6A and mfSlc26a6B cRNAs, and their pHi values were monitored in response to exposure to Cl–-free medium (Fig. 4, A, C, and D, Table 2). Compared with controls, oocytes were slightly acidified on exogenous expression of mfSlc26a6A, but not mfSlc26a6B (Table 2, initial pHi). Vm of mfSlc26a6A and mfSlc26a6B were more positive than Vm of control oocytes (Table 2, initial Vm). Exposure to 5% CO2/33 mM HCO3– acidified oocytes as a result of CO2 entry by diffusion. In oocytes expressing mfSlc26a6A and mfSlc26a6B, Cl– removal caused marked cytosolic alkalinization and hyperpolarization, which was reversed when the oocytes were returned to Cl–-containing medium (Fig. 4, C and D, Table 2, dpHi/dt and dVm). Control oocytes did not show such responses on sequential removal and addition of Cl– (Fig. 4A). Alkalinization rate and hyperpolarization were 2.3 times higher and 1.3 times lower, respectively, in mfSlc26a6A than mfSlc26a6B oocytes (Table 2, dpHi/dt and dVm). These results are similar to those of mouse Slc26a6 experiments under similar conditions (23, 52) and indicate that mfSlc26a6A and mfSlc26a6B mediate electrogenic Cl–/nHCO3– exchange (Table 2).


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  		Table 2. Calculation of activity of mfSlc26a6A and mfSlc26a6B and comparison with activity of mouse Slc26a6

 
In situ hybridization and immunohistochemical localization. Immunohistochemistry (Fig. 5) and in situ hybridization (Fig. 6) revealed an identical distribution pattern for the mRNAs of mfNBCe1, mfSlc26a6A, and mfSlc26a6B and their protein products in the epithelial cells of SW mefugu anterior intestine. By immunohistochemistry, relatively strong signals were observed in almost all epithelial cells in segment a (defined in Fig. 1B). In higher-magnification images (Fig. 5A, c, f, and i), basolateral localization of mfNBCe1 and apical membrane localization of mfSlc26a6A and mfSlc26a6B are evident. This polarized localization of the HCO3– transporters was much more clearly demonstrated by immunofluorescence microscopy (Fig. 5B). Antisera specificity was established by specific staining of mammalian culture cells (COS-7) exogenously expressing the antigens (see Fig. S1 in the online version of this article). The results of in situ hybridization were consistent with those of immunohistochemistry, showing positive signals in columnar epithelial cells (Fig. 6).


Figure 5
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  		Fig. 5. Immunohistochemistry of mfNBCe1, mfSlc26a6A, and mfSlc26a6B in anterior intestine of SW-acclimated mefugu. A: serial sections from segment a of intestine stained (brown) with anti-mfNBCe1 (a–c), anti-mfSlc26a6A (d–f), and anti-mfSlc26a6B (g–i). c, f, and i: high-magnification images areas enclosed in boxes in b, e, and h, respectively. Scale bars, 100 µm (low-magnification images: a, b, d, e, g, and h) and 10 µm (high-magnification images). Nuclei were stained (blue) by hematoxylin. B: immunofluorescence microscopic images of basolateral localization of mfNBCe1 and apical localization of mfSlc26a6 (green). Nuclei were stained by TO-PRO3 (red). Scale bars, 50 µm (low-magnification images: left) and 10 µm (high-magnification images: right).

 

Figure 6
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  		Fig. 6. In situ hybridization of mfNBCe1, mfSlc26a6A, and mfSlc26a6B in SW mefugu anterior intestine. Serial sections were obtained from segment a of intestine and stained with sense (A, D, and G) and antisense (B, E, and H) probes (purple) and counterstained with Kernechtrot for the nuclei (pink). C, F, and I: high-magnification images.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
To maintain body fluid balance, marine teleosts obtain water by drinking SW and excreting electrolytes via one of two mechanisms: 1) ions are absorbed by the intestine and excreted into the environment by the branchial chloride cells (Na+ and Cl–) or into the urine by the kidney (e.g., Mg2+ and SO42–); or 2) ions are precipitated within the intestinal fluid and rectally excreted (Ca2+ and Mg2+). Therefore, the intestine is the primary organ of body fluid balance for marine teleosts. Here, we identified HCO3– transporters that belong to the Slc4 and Slc26 families by database mining of the Takifugu genome, identified transporters that are induced during SW acclimation by Northern blot analyses, and characterized their function and localization in oocyte electrophysiology and immunohistochemistry. These HCO3– transporters are expressed on the apical and basolateral membrane of intestinal epithelial cells and upregulated during SW acclimation. We also propose a molecular model of intestinal HCO3– secretion that forms CaCO3 and MgCO3 deposits to facilitate passive water acquisition across the intestinal epithelium (Fig. 7). The model is similar to that proposed on the basis of the previous physiological measurements (5, 10–12, 14) as follows. It has been demonstrated that the intestinal HCO3– secretion by marine teleosts is dependent on luminal Cl–, suggesting that HCO3– secretion is mediated by the apical Cl–/HCO3– exchanger (5, 14, 15, 49). The serosal HCO3– and Na+ levels have also been shown to be related to intestinal HCO3– secretion (5), suggesting involvement of the basolateral Na+-HCO3– cotransporter in uptake of HCO3– from the blood. Consistent with these physiological observations, upregulation of intestinal NBCe1 expression was observed in 65% SW-acclimated rainbow trout (12). With these physiological backgrounds and in an attempt to establish the mechanism of HCO3– secretion at the molecular level, we performed a comprehensive molecular cloning analysis and Northern blot analyses and arrived at the model. Although the model is not new, the present study may be significant in providing molecular evidence for the model and in showing that the model represents a major mechanism operating in the intestine of marine teleost fish.


Figure 7
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  		Fig. 7. Model of intestinal HCO3– excretion system in SW mefugu. HCO3– transporters mfNBCe1 and mfSlc26a6 are located in basolateral and apical membranes, respectively, of anterior intestine. Driven by the Na+ gradient created by Na+-K+-ATPase, basolateral mfNBCe1 transports HCO3– into cells. Negative membrane potential generated by Na+-K+-ATPase and high Cl– concentration in intestinal fluid acts as driving force for apical HCO3– secretion by mfSlc26a6. A negative membrane potential may also act as a driving force for basolateral Cl– secretion by Cl– channel, which is not identified and illustrated. This model is similar to that proposed by other research groups on the basis of physiological analyses (5, 10–12, 14). Role of Na+-K+-ATPase in basolateral membrane of intestinal epithelial cells has been demonstrated by physiological and immunohistochemical analyses (11, 12). BBM, brush border membrane; TJ, tight junction.

 
It has been demonstrated that CA-catalyzed hydration of CO2 within the intestinal epithelium also provides HCO3– for secretion by apical Cl–/HCO3– exchangers (10, 11, 14, 49). However, our Northern analyses revealed relatively low levels of expression of any CA genes identified in the genome sequence and failed to detect significant induction of these genes in SW mefugu intestine (data not shown), suggesting that, in the case of mefugu, the contribution of CA is low and basolateral mfNBCe1 is the major pathway for supplying HCO3– to intestinal epithelial cells from the blood. This apparent discrepancy is not surprising, since the contribution of CA seems to vary greatly depending on the species (5, 10, 11, 14, 50).

The apical Na+-Cl– cotransporter (NCC) and Na+-K+-2Cl– cotransporter (NKCC) mediate Na+, K+, and Cl– uptake by the intestinal epithelium, as well as Cl– uptake by the apical anion exchanger (5, 10, 34). NKCC2β (Slc12a1β) and NCCβ (Slc12a3β) (8) are highly expressed in the eel intestine and NKCC2β expression is upregulated during SW acclimation, indicating that the Slc12 family is important in intestinal salt absorption during SW acclimation. According to this salt gradient, water in the intestinal lumen is absorbed through paracellular and transcellular routes. It has been demonstrated that expression of aquaporin 1, a water channel, is markedly elevated in the intestine of SW eels (6) or FW eels treated with cortisol (30), an SW-adapting hormone, suggesting that aquaporin 1 plays an important role in the transcellular absorption of water. Aquaporin may also be involved in volume regulation of intestinal epithelial cells. Therefore, the main physiological role of aquaporin 1 (transcellular water transport or cell volume regulation) and the contributions of the paracellular and transcellular routes to intestinal water absorption remain to be determined.

In mammals, kidney NBCe1 (kNBCe1) and pancreatic NBCe1 (pNBCe1) HCO3–-to-Na+ stoichiometry of 3:1 and 2:1, respectively (16, 43), indicates that kNBCe1 is absorptive and pNBCe1 is secretory (16, 18, 40, 42, 43). In mefugu intestine, the pancreatic type, with a putative secretory function, is expressed. NBCe1 is always localized on the basolateral membrane of epithelial cells as a result of a COOH-terminal motif (26) that is homologous in mefugu NBCe1 [QEPMLG (aa residues 1061-1066) vs. human QQPFLS (aa residues 1010–1015)]. The Phe1013 residue, conserved in mammalian NBCe1, is also essential for basolateral targeting (26). The corresponding mefugu sequence QEPMLG suggests that methionine, another bulky amino acid residue, can play a role similar to that of phenylalanine in targeting.

The rate of HCO3– secretion is proportional to the concentration of Ca2+ entering the intestine (50, 51), which is detected via extracellular Ca2+-sensing receptors (CaRs) in the apical membrane of intestinal epithelial cells (50). CaRs have been cloned from osmoregulatory tissues in the sea bream Sparus aurata (9), the dogfish shark Squalus acanthias (35), and the Mozambique tilapia Oreochromis mossambicus (28). CaR expression in the intestine is elevated in SW tilapia, and intracellular CaR signaling involves activation of phospholipase C and mitogen-activated protein kinase cascades (29). Much work remains concerning the sensing mechanisms for luminal Ca2+ concentration.

Slc26 is a family of anion exchangers that transport SO42–, Cl–, I–, formate, oxalate, OH–, and HCO3– (33). The family consists of 11 genes, which are expressed on apical (Slc26a2, Slc26a3, Slc26a4, Slc26a6, and Slc26a9) (17, 22, 27, 32, 38) and basolateral (19, 27) epithelial membranes. We demonstrated that mfSlc26a6A is strongly upregulated in the anterior intestine during SW acclimation and mfSlc26a6B is strongly expressed in the intestine of SW and FW mefugu (Fig. 2A). In mammals, Slc26a6 mediates electrogenic Cl–/nHCO3– exchange, as demonstrated using Slc26a6-expressing Xenopus oocytes and HEK-293 cells, which showed hyperpolarization of membrane potential and alkalinization of pHi when extracellular Cl– was removed from HCO3–-containing medium (23, 52). We demonstrated that mfSlc26a6A and mfSlc26a6B also mediate Cl–/nHCO3– exchange after oocyte expression. It was surprising to find that hyperpolarization of mfSlc26a6A and mfSlc26a6B is 2.0 and 2.7 times higher than in mouse Slc26a6, and the alkalinization rate of mfSlc26a6A and mfSlc26a6B is 3.1 and 1.4 times higher than that of mouse Slc26a6 (52), despite similar experimental conditions. Both mfSlc26a6A and mfSlc26a6B are localized at the apical membrane of intestinal epithelium (Fig. 5). The apical localization, activity, and induced expression of mfSlc26a6A and mfSlc26a6B in SW coincide perfectly with the requirements for intestinal Cl– absorption and HCO3– secretion, suggesting that mfSlc26a6A and mfSlc26a6B are the major apical HCO3–-secreting transporters responsible for the formation of carbonate precipitates. The anterior intestine of marine teleosts contains 60–200 mM Cl– (51), which is maintained by continuous SW drinking, despite low levels of intracellular Cl–, which can provide a Cl– gradient to drive Cl– absorption and HCO3– secretion by mfSlc26a6A and mfSlc26a6B. mfSlc26a6B is also expressed in the intestine of FW mefugu and may have a role in fluid secretion for digestion, rather than osmoregulation (45, 46).

The strong induction of mfNBCe1 at the basolateral membrane of the intestinal epithelium in SW suggests that the main source of HCO3– is the blood. Because the intestinal fluid of marine teleosts contains a high concentration of Cl– and mfSlc26a6A and mfSlc26a6B require a Cl– gradient, the amount of intracellular HCO3–, i.e., HCO3– uptake by mfNBCe1, may be the rate-limiting step. Further analysis is required to clarify the mechanism by which transporter activity is regulated.

Perspectives and Significance

In the HCO3– secretory system of the human pancreatic duct, there is a direct interaction between the STAS domain of Slc26 anion transporters and the R domain of CFTR (24) that is mediated by PKA-dependent phosphorylation of the R domain and stimulates Slc26 transporters. A similar interaction may also occur in the mefugu intestinal epithelial cell, since the STAS and R domains and the PKA consensus sequences are conserved in the mefugu Slc26a6A and Slc26a6B and CFTR (accession no. AB362833). However, the signaling that leads to intracellular accumulation of cAMP may be different, since one is for regulation of the digestive system and the other is for osmoregulation of marine teleosts. Staniocalcin, a hormone for maintaining Ca2+ homeostasis, especially in SW, where Ca2+ concentration (~10 mM) is much higher than in serum (~2 mM), may also play a role. Carbonate precipitation plays an important role not only in osmoregulation, but also in Ca2+ homeostasis, by minimizing the potential for excessive Ca2+ entry (51).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Ministry of Education, Culture, Sport, Science, and Technology of Japan (MEXT) Grants-in-Aid for Scientific Research 14104002, 17570003, and 18059010 and the 21st Century and Global Center of Excellence Program of MEXT. Work in M. F. Romero's laboratory was supported by National Eye Institute Grant EY-017732 and Cystic Fibrosis Foundation Grant Romero-06G0.


   ACKNOWLEDGMENTS
 
We thank Takeru Nakazato for discussion, Heather L. Holmes for technical support, and Setsuko Sato for secretarial assistance.

Present address: T. Nakada, Department of Molecular Pharmacology, Shinshu University School of Medicine, Nagano, Japan; A. C. Mistry, Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Hirose, Dept. of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan (e-mail: shirose{at}bio.titech.ac.jp)

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.


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S. K. Parks, M. Tresguerres, and G. G. Goss
Cellular mechanisms of Cl- transport in trout gill mitochondrion-rich cells
Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2009; 296(4): R1161 - R1169.
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