INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

FISH CAN NORMALLY SURVIVE only in neutral or close-to-neutral water. A few exceptions are the Lake Magadi tilapia Oreochromis alcalicus grahami, the only fish in the alkaline lake (pH 10) (51), the Amazonian tambaqui Colossoma macropomum, which migrates between circumneutral water and dilute acidic blackwater of the Rio Negro (70, 71), and the Osorezan dace (35). The Osorezan dace Tribolodon hakonensis, a cyprinid teleost, lives and grows in the extremely acidic (pH 3.4-3.8) Lake Osorezan and migrates to neutral streams for spawning. Lake Osorezan (formally Lake Usoriyama) is located in the Shimokita Peninsula in the northern part of Honshu, the mainland of Japan. The mechanism of adaptation of the Lake Magadi tilapia to the alkaline condition is well understood (33, 51), but the mechanism of acid adaptation of the Amazonian tambaqui and Osorezan dace has not been clarified at the molecular level.

The gill epithelium comprises five types of cells (pavement cells, mucous cells, neuroepithelial cells, undifferentiated cells, and chloride cells) and is the site of respiratory gas exchange and ion regulation (18, 26, 34, 44, 68). The gill is also believed to possess a mechanism for preventing metabolic acidosis and alkalosis (7, 8, 18). Long-term exposure of teleost fish to acidic water affects the number, distribution, and morphology of chloride cells in the gill (29, 30, 65) and can result in acute acidification of plasma and loss of NaCl, eventually leading to death. However, in Osorezan dace, these initial effects on plasma pH and Na⁺ concentration are rapidly corrected, allowing the fish to survive (25). To clarify the molecular mechanism underlying the acid tolerance, we considered it useful to identify proteins with expressions that are markedly increased in acid-adapted Osorezan dace and performed subtraction cloning. Among several clones isolated from a cDNA library enriched in acid-inducible messages, there was a clone encoding the Na⁺- and K⁺-dependent ATPase (Na⁺-K⁺-ATPase), the Na⁺ pump that generates the driving force for other ion-transporting systems, and surprisingly its message level was markedly increased in the gill, but not in the kidney, of acid-adapted Osorezan dace. These observations, together with the life history and acid tolerance of the animal, suggested that chloride cells in the gill of Osorezan dace play an important role in maintaining acid-base balance in a pH 3.5 environment by excreting H⁺ and transporting HCO into the blood for neutralization of plasma and that these functions are supported by the driving force generated by the Na⁺-K⁺-ATPase. We further hypothesized that carbonic anhydrase (CA), which catalyzes the reversible hydration of CO₂ (CO₂ + H₂O  H⁺ + HCO) and in mammals contributes to control of pH and ion transport in the stomach, pancreas, and kidney (56), is also required for these processes in Osorezan dace. We therefore cloned the cDNAs for CA and candidate H⁺ and HCO transporters from this fish species and determined the localization and regulation of expression of the corresponding genes. Our results indicate that a type II CA (CA-II), a type 1 Na⁺-HCO cotransporter (NBC1), and a type 3 Na⁺/H⁺ exchanger (NHE3) are indeed highly expressed, with the expected polarities, in chloride cells of the acid-adapted Osorezan dace, compared with the low levels of expression apparent in fish acclimatized to neutral water.

This regulatory system is somewhat reminiscent of that working in the mammalian renal proximal tubule cells to defend against metabolic acidosis by reabsorbing bicarbonate and returning it to the buffer pool of the body (20, 61). Other important mechanisms operating in the proximal tubules are ammoniagenesis and gluconeogenesis, which generate two ammonia and two bicarbonate from the metabolism of glutamine and facilitate the excretion of acids and partially restore normal acid-base balance (6, 10). We therefore further examined the contribution of glutamine catabolism to systemic pH homeostasis and found that expression of glutamate dehydrogenase (GDH), a mitochondrial matrix enzyme that catalyzes oxidative deamination of glutamate and enhances ammonia production, is also markedly elevated in virtually all tissues when Osorezan dace is faced with acid challenges, suggesting a significant role in the acid adaptation. On the basis of these observations, we proposed a mechanism in which 1) the compensatory system in the chloride cells plays a major role in overcoming the Na⁺ loss and acidification and 2) glutamine catabolism in the whole body accomplishes a fine adjustment of systemic pH of Osorezan dace in a pH 3.5 lake.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Materials. The Osorezan dace were caught by netting in a neutral stream during the spawning period (June) and acclimated in a 1-ton freshwater tank for >1 mo before use. The water chemistry was as follows: pH 3.5-3.7, 0.92 mM Na⁺, 0.84 mM Cl, 0.06 mM K⁺, 0.21 mM Ca²⁺, 0.07 mM Mg²⁺, and 0.49 mM SO for lake water and pH 6.8-7.2, 0.40 mM Na⁺, 0.25 mM Cl, 0.02 mM K⁺, 0.11 mM Ca²⁺, 0.05 mM Mg²⁺, and 0.03 mM SO for neutral stream water. Control dace (25) were obtained from the Freshwater Fisheries and Environment Division, National Research Institute of Fisheries Science (Ueda, Nagano, Japan). Blood sampling and measurements of blood pH and plasma concentration of Na⁺ were performed as described previously (25).

Transfer experiment. To examine the time-course changes in the mRNA levels of vacuolar-type H⁺-translocating ATPase (V-ATPase) B subunit, Na⁺-K⁺-ATPase, CA-II, NHE3, NBC1, aquaporin-3 (AQP3), and GDH after transfer to acidic water, neutral water-adapted dace (n = 40) were transferred directly to acidic lake water, and the gills were sampled from 10 dace on days 0, 1, 2, 5, and 7 for Northern blot analysis.

Molecular cloning. Fragments of B subunit of V-ATPase, CA-II, NHE3, NBC1, and GDH cDNAs were isolated from Osorezan dace by RT-PCR with gill mRNA. The degenerate primers were as follows: 5'-ATIGCIGCICARATHTG-3' (sense) and 5'-ATITCITCRTTNGGCAT-3' (antisense) for B subunit of V-ATPase, 5'-CARTTYCAYTTYCAYTGGGG-3' (sense) and 5'-SRTAYTTNGTRTTCCARTG-3' (antisense) for CA-II, 5'-GCNGTKCTGGCNGTITTYGARGA-3' (sense) and 5'-GCNCCNCKNARICCICCRTA-3' (antisense) for NHE3, 5'-GARAARGTNGTRAARGGNGG-3' (sense) and 5'-TGRAANACYTCRTCISWCAT-3' (antisense) for NBC1, and 5'-ATGACNTAYAARTGYGCNGT-3' (sense) and 5'-GCRTANGTRTCNGCDATCCA-3' (antisense) for GDH, where R is A or G, Y is C or T, S is C or G, N is C, G, A, or T, K is G or T, W is A or T, D is A, G, or T, H is A, C, or T, and I is inosine. Fragments of cDNAs for the -subunit of Na⁺-K⁺-ATPase and for AQP3 were isolated with a subtraction cloning kit (Clontech); corresponding full-length clones were obtained by screening a dace gill cDNA library constructed in ZAP II (Clontech). Filters were prehybridized in a solution containing 50% formamide, 5× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 5× Denhardt's solution (1× Denhardt's is 0.2% Ficoll, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin), and 0.1% SDS for 1 h at 42°C and then hybridized overnight at 42°C in the same solution containing [-³²P]dCTP-labeled cDNA probe. Filters were washed in 1× SSC and 0.1% SDS at 60°C for 5 h. Positive inserts were subcloned into pBluescript II (Stratagene) by the in vivo excision and recircularization process in accordance with the manufacturer's protocol and sequenced.

Northern blot analysis. At various times after acid (pH 3.5) adaptation of Osorezan dace, we isolated total RNA from the pools of various tissues of 10 fish and subjected it to Northern blot analysis with a Hybond-N⁺ membrane (Amersham Biosciences) and [-³²P]dCTP-labeled cDNA probes. Hybridization was performed in PerfectHyb solution (Toyobo) at 68°C for 20 h. The membrane was then washed and exposed to an imaging plate, which was processed with a Fujix BAS 2000 bioimaging analyzer. The probes comprised nucleotides 1-480 of V-ATPase B subunit cDNA, nucleotides 315-415 of CA-II cDNA, nucleotides 258-1147 of NHE3 cDNA, nucleotides 767-1403 of NBC1 cDNA, nucleotides 71-679 of AQP3 cDNA, nucleotides 1-173 of GDH cDNA, and nucleotides 1-370 of the Na⁺-K⁺-ATPase -subunit cDNA. RNA molecular weight marker II (1.5-6.9 kb; Roche) was used as the size marker.

Cell culture and cDNA transfection. PS120 cells (47) were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 7.5% (vol/vol) fetal calf serum at 37°C in an atmosphere of 95% air-5% CO₂. The cloned dace NHE3 (dNHE3) and dace NBC1 (dNBC1) cDNA were introduced into the pECE vector and transfected into PS120 cells (5 × 10⁵ cells/10-cm dish) by means of the calcium phosphate coprecipitation technique. Cell populations that stably express dNHE3 and dNBC1 were selected by means of the repetitive H⁺-killing selection procedure (62). In the case of dNBC1, recovery medium contained 2 mM NaHCO.

Measurement of ²²Na+ uptake for dNHE3 activity. The rates of ethylisopropylamiloride (EIPA)-sensitive ²²Na⁺ uptake by PS120 cells expressing dNHE3 were measured using cells clamped at intracellular pH (pH_i) 5.6 by the standard K⁺/nigericin method. For measurement of ²²Na⁺ uptake, cells were preincubated for 1 h in NH₄Cl medium (50 mM NH₄Cl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM KCl, 70 mM choline chloride, 5 mM glucose, and 15 mM HEPES-Tris, pH 7.4), washed twice with choline chloride medium (1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM KCl, 120 mM choline chloride, 5 mM glucose, and 15 mM HEPES-Tris, pH 7.4), and then incubated for 15 min in the same medium additionally containing 1 mM ²²NaCl (370 kBq/ml), 1 mM ouabain, and various concentrations of EIPA. Cells were washed four times with ice-cold, nonradioactive choline chloride medium, and then ²²Na⁺ radioactivity was counted.

pH_i measurement for dNBC1 activity. Changes in pH_i were monitored using the acetoxymethyl ester of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM), as described previously (2, 58, 59). Transfected cells were grown to 70-80% confluent density on coverslips and serum starved for 10 h to arrest growth. At the start of the experiment, the culture medium was removed, incubated in the presence of 2 µM BCECF in a choline chloride medium for 5 min, and washed to remove extracellular dye. The coverslip was mounted in a cuvette, and cells were pulsed in NH₄Cl medium in the presence of 30 mM Na⁺ or 30 mM HCO for 8 min. Removal of NH₄Cl and perfusion with choline chloride medium resulted in stable acidification of the cells. The cuvette was then perfused with 30 mM Na⁺- and 30 mM HCO-containing medium, and pH_i recovery was measured. pH_i was measured in a thermostatically controlled holding chamber (37°C) in a Delta Scan dual-excitation spectrofluorometer (Photon Technology International, South Brunswick, NJ). The fluorescence ratio at excitation wavelengths of 500 and 450 nm was utilized to determine pH_i values in the experimental groups by comparison with the calibration curve that was generated by the standard KCl/nigericin technique.

Xenopus oocyte electrophysiology for dNBC1 activity. dNBC1 cDNA (4.5 kb) including untranslated regions was subcloned into a Xenopus oocyte expression vector, pGEMHE. The resulting plasmid was linearized with NheI and then used as a template for cRNA synthesis from the T7 promoter using mMessage mMachine (Ambion, Austin, TX). Fifty nanoliters of dNBC1 cRNA (0.5 µg/µl) or water (control) were injected into collagenase-dissociated, stage V-VI Xenopus oocyte, as previously described (53). On days 3-10 after cRNA injection, transport activity was studied using pH_i and voltage microelectrodes in a constant-perfusion chamber, as described elsewhere (53). pH electrodes were calibrated with traceable pH 6.0 and 8.0 solutions and had slopes of about 56 mV/pH unit. Oocyte solutions were those used previously (53) titrated to pH 7.5 and ~200 mosM. CO₂/HCO solutions were bubbled continuously with 1.5% CO₂, resulting in 10 mM HCO, pH 7.5. Choline was used to replace Na⁺ for the "0 Na⁺" solution.

Immunohistochemistry and immunofluorescence. Ten dace were acclimated in neutral water for 2 wk and then transferred to acidic water. On day 5 after transfer, gills were removed and fixed for 2 h in PBS containing 4% (wt/vol) paraformaldehyde at 4°C. After fixation, they were separated into two groups and embedded in Tissue Tek OCT compound or in paraffin. Frozen sections (6 µm) and paraffin-embedded sections (4 µm) were prepared, immersed in PBS containing 1% H₂O₂, exposed to 2% normal goat serum, and incubated with affinity-purified primary antibodies. Primary antibodies were generated in rabbits in response to injection with B subunit of dace V-ATPase (a partial 160-residue recombinant protein fused to a histidine tag), dace CA-II (a full-length 260-residue recombinant protein fused to a histidine tag), dNHE3 (a 20-residue COOH-terminal synthetic peptide linked to keyhole limpet hemocyanin: DASVDEEASEEKPGKNHTRL), NBC1 (a 42-residue COOH-terminal fragment fused to a histidine tag: SDFPAIENVPSIKISMETMEQDPVLGEKPSDRNKPMSFLTPY), or AQP3 (a 20-residue COOH-terminal synthetic peptide linked to keyhole limpet hemocyanin: DKTNKDMEESLKLNDVTGKN), and they were purified on HiTrap NHS-activated HP columns (Amersham Biosciences) containing the corresponding immobilized antigen. The antibodies were used at dilutions of 1:3,000 (anti-CA-II), 1:32,000 (anti-NHE3), 1:10,000 (anti-NBC1), or 1:3,000 (anti-AQP3). For staining of Na⁺-K⁺-ATPase, an antiserum to the eel protein that cross-reacts with the dace protein was used (36). After the sections were washed in PBS, they were incubated with peroxidase-conjugated secondary antibodies (Dako), and the peroxidase was detected with diaminobenzidine.

Western blot analysis. Neutral water and acidic water dace gills were homogenized in 100 mM Tris buffer containing 0.9% NaCl, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin, and 10 mg/ml leupeptin. The homogenates were centrifuged at 5,000 g for 20 min, and the pellets were resuspended in the same buffer. These procedures were repeated three times at 4°C. The membrane proteins were separated by SDS-PAGE and transferred to Immobilon polyvinylidene difluoride membrane (Millipore). After the membrane was blocked in Tris-buffered saline with Tween 20 [150 mM NaCl, 0.05% (vol/vol) Tween 20, and 10 mM Tris · HCl, pH 8.0] containing 5% (vol/vol) nonfat milk for 1 h at room temperature, it was incubated with anti-B subunit of V-ATPase antiserum at 1:3,000 dilution for 10 h at 4°C. After it was washed, the membrane was incubated for 1 h at room temperature with an alkaline phosphatase-conjugated mouse anti-rabbit IgG antibody (Sigma) diluted 1:5,000 in Tris-buffered saline-Tween 20. The membrane was then developed with 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate and 0.4 mM nitro blue tetrazolium chloride (Wako Pure Chemicals) in 0.1 mM Tris · HCl, pH 9.5, containing 50 mM MgCl₂ and 150 mM NaCl.

In situ hybridization. In situ hybridization histochemistry was performed with paraffin-embedded sections (4 µm) according to the protocol recommended by the manufacturer of a digoxigenin RNA labeling kit (http://biochem.roche.com/biochemica/no1_98/p10.pdf). Sections were subjected to hybridization with a digoxigenin-labeled cRNA probe of 315 bp that is complementary to the mRNA sequence encoding a central portion of CA-II that includes the signature sequence; the corresponding 315-bp sense probe was used as a control.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Extraordinary abilities of Osorezan dace to prevent acidification and loss of Na+. Previous physiological studies have demonstrated remarkable acid tolerance of the Osorezan dace (25, 35). We confirmed this by measuring changes of blood pH and plasma Na⁺ after transfer of the Osorezan dace from neutral to acidic (pH 3.5) water and by comparing them with those of control dace (Fig. 1). The blood pH and plasma Na⁺ concentration of the control dace declined continuously in the acidic environment, and most of them died within 36 h. In contrast, in the case of the Osorezan dace, the blood pH and Na⁺ concentration, which were once lowered, were restored to close-to-normal levels in 24 h, and they all survived (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Changes in blood pH (A) and plasma Na+ concentration ([Na+], B) during adaptation to an acidic environment. Changes in blood pH and plasma [Na+] in Osorezan dace () and control Ueda dace () were measured after their transfer from neutral (pH 6.8-7.2) to acidic (pH 3.5) water. Survival rate of Osorezan dace was 100% when they were transferred from neutral water to water at pH 3.5, whereas only 66% of control dace survived for >24 h, and most of them died within 36 h under such acidic conditions. Values are means ± SE from 7-11 fish. *Significant differences between 2 groups of dace, P < 0.05 (Student's t-test). #Significantly different from initial values within the same species, P < 0.05.

Relatively small changes in V-ATPase levels. The gills of the fish are the primary site of ionic and acid-base regulation. The favored model proposes that an apically oriented V-ATPase plays a major role in acid-base regulation and uptake of Na⁺ from the environment by excreting acid (H⁺) and providing a driving force to apical membrane Na⁺ channels (32, 45, 46). To begin to understand the molecular mechanisms of the acid tolerance of the Osorezan dace, the expression levels of V-ATPase were examined using a specific cDNA probe and antiserum with the expectation that if it is a key molecule for acid adaptation, its levels would be greatly elevated in the gill when the dace is transferred from neutral to acidic water.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Significant but relatively low contribution of vacuolar-type H+-translocating ATPase (V-ATPase) to acid adaptation of Osorezan dace. A: time course of mRNA expression of B subunit of V-ATPase in the gill after transfer of Osorezan dace to pH 3.5 water. Northern blot analysis was performed with 20 µg of total RNA, which revealed a single band at 2.6 kb. Exposure time (4 days) was much longer than for Na+-K+-ATPase, type II carbonic anhydrase (CA-II), type 3 Na+/H+ exchanger (NHE3), type 1 Na+-HCO cotransporter (NBC1), aquaporin-3 (AQP3), and glutamate dehydrogenase (GDH), i.e., 6-12 h (see Figs. 4 and 10). B: Western blot analysis of B subunit of V-ATPase in gills of dace adapted to neutral and acidic water. A specific antiserum was raised against recombinant B subunit of dace V-ATPase. Antiserum recognized a band at 55 kDa; size of this band is consistent with that reported for B subunit of other species (14, 15).

Marked elevation of Na+-K+-ATPase mRNA in the gill but not in the kidney. In a parallel attempt, we tried to identify genes that are highly expressed in acid-adapted Osorezan dace by constructing a subtraction cDNA library from mRNA preparations of the gills of the Osorezan dace acclimated to neutral and pH 3.5 water. Sequencing of cDNA clones of the subtraction library indicated the presence of Na⁺-K⁺-ATPase (DDBJ/EMBL/GenBank accession no. AB056155) in a relatively high proportion, suggesting that the Na⁺-K⁺-ATPase message is highly elevated in the gill of acid-adapted Osorezan dace. Indeed, Northern blot analysis indicated a marked induction of Na⁺-K⁺- ATPase mRNA in the gill on acidification (Fig. 3). However, its levels in the kidney, one of the richest sources of Na⁺-K⁺-ATPase, were not significantly affected by acidification (Fig. 3B). This marked induction of branchial Na⁺-K⁺-ATPase mRNA, together with the accumulating evidence for the role of the fish gill in ion transport beginning with Smith's study in 1930 (57), provided the initial clue that the ion transport systems in the gill might be responsible for the acid tolerance of the Osorezan dace, because Na⁺-K⁺-ATPase has been demonstrated to have an important role in driving a variety of ion-transporting processes. We believed that CA and the downstream ion transporters that have long been recognized to participate in the control of pH in a variety of tissues including the branchial epithelium (21, 69) may play major roles and initiated the studies described below.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Abundance of Na+-K+-ATPase mRNA in gill of Osorezan dace. Northern blot analysis was performed to determine time course of induction (A) and abundance of Na+-K+-ATPase mRNA in gill (A and B) or kidney (B) of Osorezan dace after transfer of fish to pH 3.5 water. Size of hybridizing band is ~3.0 kb. Total RNA (20 µg) was also subjected to hybridization with a -actin cDNA probe to confirm equal loading of samples.

CA-II levels and locations. A CA cDNA probe was obtained by RT-PCR performed with Osorezan dace gill mRNA and a set of primers based on the highly conserved regions of CAs cloned previously. A full-length cDNA was then isolated from a dace gill cDNA library. Sequence analysis indicated that the cDNA encodes CA-II, which is composed of 260 amino acid residues (DDBJ/EMBL/GenBank accession no. AB055617; Fig. 4A); such isoforms are cytosolic and possess the highest catalytic activity of the 11 different active isoforms of CA identified to date, which include cytoplasmic, mitochondrial, and membrane-bound forms (38, 56). CA-II is expressed in a wide variety of mammalian tissues. However, Northern blot analysis revealed that CA-II mRNA was present in substantial amounts only in the gill and anterior intestine of acid-exposed Osorezan dace; RNA preparations from these organs of control fish maintained in neutral water yielded only a faint hybridizing band under the same conditions (Fig. 4C). Transfer of fish to acidic water resulted in a marked induction of CA-II mRNA in the gill within 1 day (Fig. 4B). Immunohistochemistry with gill sections revealed that CA-II is localized to the chloride cells (see Fig. 8, A and B). In situ hybridization demonstrated that CA-II mRNA is highly expressed in the chloride cells of the gill (data not shown).

View larger version (71K): [in this window] [in a new window]   Fig. 4.   Cloning, tissue distribution of transcripts, and acid-induced changes in expression of CA-II (A-C), NHE3 (D-F), NBC1 (G-I), and AQP3 (J-L) genes. A, D, G, and J: schematic representations of structures and membrane topologies of CA-II, NHE3, NBC1, and AQP3 proteins predicted from cDNAs cloned from Osorezan dace. Arrows in A indicate Zn2+-binding sites; filled box represents signature sequence (SEHTVDGKCYPAELHLV) for eukaryotic CA. Lines delineated by arrowheads in A, D, and G indicate parts of sequences that were amplified by degenerate RT-PCR.  In D indicates a PDZ-binding motif (HTRL) at COOH terminus of NHE3.  In J represent NPA motifs. Numbers indicate NH2- and COOH-terminal residues. Overall sequence identities of dace CA-II, NBC1, and AQP3 to their human counterparts are 64, 80, and 68%, respectively. Dace NHE3 is 61% identical to human NHE3 in its 12 membrane-spanning domains but is only 31% identical in intracellular COOH-terminal region. B, E, H, and K: time courses of abundance of respective mRNAs in gill after transfer of Osorezan dace to pH 3.5 water. C, F, I, and L: tissue distribution of respective mRNAs before (day 0) and 7 days after transfer of fish to acidic water. Ant, anterior; post, posterior. Northern blot analysis was performed with 20 µg of total RNA. Sizes of hybridizing bands are ~2.0 kb (CA-II), 4.0 kb (NHE3), 4.5 kb (NBC1), and 2.9 kb (AQP3).

Cloning and Northern analysis of NHE3 and NBC1. Similar approaches were adopted to identify candidates for the proteins that mediate the transport of H⁺ and HCO, yielding cDNAs that encode the A and B subunits of V-ATPase, three isoforms of NHE (NHE1-NHE3), and an NBC. Among the transcripts encoding these candidate proteins, NHE3 and NBC1 mRNAs were readily detected in the gill of acid-adapted Osorezan dace by Northern blot analysis of total RNA (Fig. 4, F and I). The amounts of NHE3 and NBC1 mRNAs in the gill were markedly increased within 1 day of transfer of fish from neutral to acidic water (Fig. 4, E and H). Whereas NHE3 mRNA was also relatively abundant in the kidney of acid-adapted fish (Fig. 4F), NBC1 mRNA was present at a high concentration only in the gill (Fig. 4I). In situ hybridization demonstrated that NHE3 and NBC1 mRNAs are localized to chloride cells in the gill (data not shown).

Structural and functional properties of dNHE3 and dNBC1. The nucleotide sequence of the full-length open reading frame of dNHE3 cDNA indicated that it encodes an integral membrane protein of 827 amino acid residues with 12 transmembrane spans (DDBJ/EMBL/GenBank accession no. AB055466; Fig. 4D). The dNHE3 shares a relatively high similarity with the mammalian counterparts in its membrane-spanning domains, but it is quite divergent in the intracellular COOH-terminal region (Fig. 4D). For determining its functional property, a cell line stably expressing dNHE3 was established by transfecting PS120 cells, a cell line lacking NHEs (47), with the expression vector pECE carrying the coding region of dNHE3, and its Na⁺/H⁺ exchange activity was measured using ²²Na⁺ (Fig. 5). The activity was seen only in the transfected cells and was sensitive to EIPA (Fig. 5A), a derivative of amiloride. Compared with mammalian NHE1 and NHE3 (63), the dace exchanger was closer to the NHE3 isoform in its amiloride sensitivity (Fig. 5B), consistent with the classification based on the sequence similarity.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Functional characterization of dace NHE3. A: initial rates of H+-activated 22Na+ influx measured in untransfected PS120 cells and in a clonal isolate (PS120dNHE3) expressing dace NHE3 in the absence or presence of 1 mM ethylisopropylamiloride (EIPA, n = 4). B: concentration-response curves for inhibition of 22Na+ uptake by EIPA in human NHE1 (hNHE1), human NHE3 (hNHE3), and dace NHE3 (dNHE3) stably expressed in PS120 cells (n = 4). hNHE1- and hNHE3-expressing PS120 cell lines have previously been established by Wakabayashi et al. (63).

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Functional characterization of dace NBC1. PS120 cells stably transfected with dace NBC1 were acid loaded by NH withdrawal and monitored for intracellular pH (pHi) recovery. Presence or addition of key ion species, for determining Na+ and HCO dependency of dace NBC1 activity, is indicated above A and B (open rectangles). After acidification, cells were exposed to an Na+-containing solution (A, n = 4) or an HCO-containing solution (B, n = 4), in the presence or absence of 300 µM DIDS.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Xenopus oocyte electrophysiology: representative pHi and membrane potential (Vm) experiments of water control (A) and dace NBC1 (B) oocytes. Solution changes are indicated by horizontal bars. Time scale is identical for both oocyte experiments. See RESULTS AND DISCUSSION for experimental details. 10 HCO, 10 mM HCO; 0 Na+, 0 mM Na+.

Immunohistochemical localization of NHE3 and NBC1. To clarify the physiological roles of NHE3 and NBC1, we investigated whether these proteins are located in the apical or basolateral membrane of chloride cells. We therefore first prepared specific antisera to the most COOH-terminal fragments (20-42-residue long) of these proteins. Immunohistochemical analysis with the resulting antibodies revealed the presence of marked NHE3 immunoreactivity on the apical side of chloride cells (Fig. 8, C and D), whereas NBC1 immunoreactivity was localized to the basolateral membrane that forms the tubular network (and thereby increases the cell surface area) by extensive infolding into the cytoplasmic space (Fig. 8E). This latter structural feature renders the staining patterns of proteins in the basolateral membrane indistinguishable from those of cytoplasmic proteins. The Na⁺-K⁺-ATPase, a marker protein of the basolateral membrane of the chloride cell (55), yielded a staining pattern similar to that of NBC1 (Fig. 8F). Their colocalization was, however, not complete; NBC1-positive cells appear to represent a subpopulation of the Na⁺-K⁺-ATPase-positive chloride cells. We therefore performed double-immunofluorescence staining for NBC1 and Na⁺-K⁺-ATPase. Indeed, a subpopulation of Na⁺-K⁺-ATPase-positive cells were stained with anti-NBC1 (Fig. 9). Also noteworthy is a follicular arrangement of chloride cells in the acid-adapted dace gill (Fig. 8D), which was not seen in the neutral water-acclimated Osorezan dace (data not shown) (25). This follicular arrangement may be one of the strategies to exclude H⁺ efficiently.

View larger version (78K): [in this window] [in a new window]   Fig. 8.   Immunohistochemical analyses of distribution of CA-II, NHE3, NBC1, and AQP3 in gill of acid-adapted Osorezan dace. Cross sections of frozen (A, B, G, and H) or paraffin-embedded (C-F) gills were cut and stained with antibodies to CA-II (A), NHE3 (C and D), NBC1 (E), or AQP3 (G). Na+-K+-ATPase was also stained with specific antibodies in serial sections (B, F, and H) corresponding to those in A, E, and G, respectively, as a marker for chloride cells. D: higher-magnification view of C showing apical staining of chloride cells in a follicular arrangement (arrowheads). Scale bars, 50 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Immunofluorescence localization of dace NBC1 and Na+-K+-ATPase in acid-adapted dace gill. Frozen gill sections were prepared from 5 acid-adapted dace, 2 of which are shown as A and B, and stained with Alexa 488-conjugated antibody to dace NBC1 (A1 and B1, green) and Cy3-conjugated antibody to Na+-K+-ATPase (A2 and B2, red). A3: merge of A1 and A2; B3: merge of B1 and B2. Nuclei were stained with Hoechst 33342 (blue). A4 and B4: gill sections corresponding to A1-A3 and B1-B3, respectively. Gray areas represent chloride cells in a follicular arrangement: light gray cells expressed only Na+-K+-ATPase; dark gray cells expressed Na+-K+-ATPase and NBC1. Arrowheads point to openings of follicular pits. Scale bars, 20 µm.

Role of NHE3. Members of the NHE family of proteins are present in the plasma membranes of virtually all animal cells, where they mediate the electroneutral exchange of intracellular H⁺ for external Na⁺ with a 1:1 stoichiometry (9, 43, 64). Seven NHE isoforms (NHE1-NHE7) have been identified in mammalian cells and share a membrane topology characterized by 12 transmembrane domains in the NH₂-terminal region and a large COOH-terminal tail located in the cytosol. The isoforms NHE1-NHE4 are expressed in epithelial cells; NHE5 is abundant in the brain; NHE6 (42) is an endoplasmic reticulum/recycling endosomal isoform (5, 37); and NHE7 is in the trans-Golgi network (41). NHE1, NHE6, and NHE7 are widely expressed and are believed to perform housekeeping functions such as the control of pH_i and maintenance of ionic homeostasis in subcellular organelles. Among the epithelial isoforms, NHE2 is preferentially expressed in the gastrointestinal tract. NHE3 (60) is confined to the apical membrane of polarized epithelial cells of the kidney (3), gastrointestinal tract (22), and fish gill (13), and it is therefore believed to perform specialized functions. The structure (Fig. 4D) and predicted function of dNHE3 are consistent with this notion. The relatively divergent COOH-terminal tail of dNHE3 suggests that a specific mechanism may be responsible for regulation of its function. Chloride cells are rich in mitochondria, are metabolically active, and contain large amounts of Na⁺-K⁺-ATPase, a membrane-bound enzyme that couples ATP hydrolysis to the exchange of Na⁺ and K⁺ with a 3:2 stoichiometry to maintain a low intracellular Na⁺ concentration (7, 26, 34, 68). This Na⁺ pump activity and the protons generated by CA-II may provide the driving force for NHE3 (see Fig. 11A). Recently, Edwards et al. (12) isolated a partial cDNA clone of NHE3 from the gills of the Atlantic hagfish and showed by RT-PCR that its message levels in the gill were elevated more than four times after an induced metabolic acidosis. In this marine fish, downhill Na⁺ influx can be used for H⁺ efflux; but in the case of Osorezan dace, such a large concentration gradient of Na⁺ is not available; furthermore, a steep (>1,000-fold) uphill H⁺ transport is necessary. Whether this problem can be overcome by strong induction alone of the driving-force providing CA-II and Na⁺-K⁺-ATPase remains to be established. The follicular arrangement of the chloride cells in acid-acclimated Osorezan dace may make some contribution by partially sealing the lumen of the follicle from low-pH lake water.

Role of NBC1. NBC mediates the coupled transport of Na⁺ and HCO across the plasma membrane and, thereby, contributes to the secretion and absorption of HCO and the regulation of pH_i (52). NBC was first identified in the salamander kidney (4) and was subsequently cloned from the same source (53). Four types of NBCs (NBC1-NBC4) have been identified in mammals (24, 49, 50). The NBC now cloned from the acid-adapted dace gill (Fig. 4G) is similar to NBC1, which transports Na⁺ and HCO with a stoichiometry of 1:2 (an electrogenic process). The electrogenic nature of NBC1-mediated ion transport (Fig. 7B) suggests that the efflux of HCO from chloride cells is stimulated not only by the supply of HCO provided by CA-II but also by the hyperpolarization of these cells caused by the sustained activity of Na⁺-K⁺-ATPase. These observations are consistent with the model proposed in Fig. 11. dNBC1 is most similar to pNBC1/NBCe1-B, which has an extended NH₂ terminus (52). Our model of dace gill transport is similar to the cellular model of mammalian proximal tubule HCO absorption. Figure 4I indicates that there is no detectable dNBC1 mRNA in the kidney, even though a probe common to NBC1 isoforms was used. Thus, at least for acidosis, the gill, rather than the kidney, is crucial for systemic acid-base homeostasis. At the molecular level, pNBC1/NBCe1-B, rather than kNBC1/NBCe1-A, is the major homeostatic HCO transporter in the Osorezan dace.

Potential role of AQP3. An attempt to identify genes that are specifically expressed in dace gill under acidic conditions by screening a subtracted cDNA library suggested that the gene for the water channel AQP3 (DDBJ/EMBL/GenBank accession no. AB055465) might be one such candidate (for the initial cloning and review of aquaporins, see Refs. 16, 23, 48). Northern blot analysis revealed that AQP3 mRNA is enriched in the gill compared with other tissues of acid-adapted dace (Fig. 4L) and that its abundance in this tissue is markedly increased within 1 day of the transfer of fish to acidic water (Fig. 4K). We therefore determined the predicted amino acid sequence of dace AQP3 (Fig. 4J), prepared antibodies to a synthetic peptide based on this sequence, and examined the expression of this protein in the gill by immunohistochemistry. Marked AQP3 staining was observed in the chloride cells (Fig. 8G) in a pattern that resembled that of Na⁺-K⁺- ATPase immunoreactivity (Fig. 8H). This observation suggests that acid-induced expression of AQP3 results in stimulation of water transport across the basolateral membrane and, thereby, provides a substrate for CA-II (see Fig. 11A). Although not illustrated in Fig. 11, the possibility also exists that AQP3 is somehow involved in osmoregulation (e.g., indirect involvement in Na⁺ uptake), because Cutler and Cramb (11) and Lignot and coworkers (31) recently demonstrated a marked downregulation of AQP3 in eel gill chloride cells after seawater acclimation. Another potential role of AQP3 may be acceleration of ammonia secretion, because its relative, AQP1, has been demonstrated to transport ammonia (40), and it is known that 1) freshwater fish excrete a higher percentage of total body ammonia across the gills than marine teleosts (66, 67) and 2) plasma ammonia levels increase in the Amazonian tambaqui on transfer from neutral to acidic water (71).

Role of glutamine catabolism. In mammals, it has been established that increased renal ammoniagenesis and gluconeogenesis (see Fig. 11B) constitute an adaptive response for restoring acid-base balance during metabolic acidosis (10). To determine whether the system plays a role in acid adaptation of the Osorezan dace, we measured induction of its component GDH (Fig. 10) by Northern blot analysis. A partial 173-bp cDNA for dace GDH exhibiting 97% sequence similarity to human GDH was obtained by RT-PCR (DDBJ/EMBL/GenBank accession no. AB094342) and used as a hybridization probe. Large inductions of GDH mRNA were observed in all tissues examined when dace were exposed to acid (Fig. 10B). This increase in ubiquitous tissues is a marked contrast to the tissue- and cell-specific increases of CA-II, NHE3, NBC1, Na⁺-K⁺-ATPase, and AQP3 descried above (Figs. 3 and 4).

View larger version (37K): [in this window] [in a new window]   Fig. 10.   Acid-induced stimulation of ammoniagenesis monitored by Northern blot analysis of GDH message. A: time course of mRNA expression in gill after transfer of Osorezan dace to pH 3.5 water. B: GDH mRNA levels in various tissues of Osorezan dace before (day 0) and 7 days after transfer of fish to acidic water. Northern blot analysis was performed with 20 µg of total RNA, and a single band of 1.9 kb was detected.

Proposed mechanism. In survival of the Osorezan dace, the combined roles of 1) CA and its upstream (AQP3) and downstream molecules (NHE3 and NBC1) in chloride cells of the gill and 2) ammoniagenesis and gluconeogenesis in various tissues are schematically shown in Fig. 11. As a coarse adjustment taking place in the chloride cell, CA-II, a Zn²⁺-containing enzyme, provides HCO and H⁺ for the regulation of pH homeostasis. Protons are excreted across the apical membrane by NHE3 in exchange of Na⁺, whereas HCO is cotransported with Na⁺ across the basolateral membrane into the blood by NBC1 (Fig. 11A). Na⁺-K⁺-ATPase, an electrogenic Na⁺ pump, provides the driving force for NHE3 and NBC1 by reducing the intracellular concentration of Na⁺ and by hyperpolarizing the cell, respectively. AQP3 may also contribute to this system by supplying H₂O to CA-II.

View larger version (29K): [in this window] [in a new window]   Fig. 11.   Two mechanisms essential for overcoming acid challenges. A: model for concerted actions of CA-II, NHE3, NBC1, AQP3, and Na+-K+-ATPase in chloride cell of Osorezan dace. Expressions of these molecules are strongly induced on acidification and reach levels that can be readily detected by Northern blot analysis using total RNA preparations. Electrogenic natures of NBC1 and Na+-K+-ATPase are represented by unequal arrow sizes. Bay-like structures of basolateral membrane represent numerous infoldings of membrane forming tubular system. For explanation of the mechanism and functional role of each component, see RESULTS AND DISCUSSION. B: generation of ammonia and bicarbonate through metabolism of glutamine, which is activated on acidification in various tissues of Osorezan dace and is expected to play a significant role in survival in acidic water. In mammals, similar induction of components of glutamine metabolism has also been well established and shown to be sensitive to small pH changes but can be seen only in renal proximal tubule cells during metabolic acidosis. Mito, mitochondria; Q, glutamine; Glnase, glutaminase; E, glutamic acid; Mal, maleic acid; OAA, oxaloacetic acid; PEPCK, phosphoenolpyruvate carboxykinase; PEP, phosphoenolpyruvate.

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