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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; 2Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan; and 3Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China
Submitted 15 January 2008 ; accepted in final form 4 March 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ionocytes; Na+/H+ exchanger; skin; gill; embryo
H2 + CO2 HCO3– + H+. This reaction forms the basis for the regulation of the acid-base balance in organisms. The 15 CA isoforms identified in mammals so far have been found to differ in their kinetic properties, susceptibility to inhibitors, and specific tissue distributions (29). CA2 is the only soluble form of carbonic anhydrase in renal epithelial cells. On the other hand, CA4 is expressed on either the apical brush-border membrane and/or the basolateral membrane of proximal tubule cells (29). The kidneys reabsorb all the bicarbonate that is filtered by the glomeruli. Most, 70%
85%, of the bicarbonate is reabsorbed in proximal tubules and only 10%20% in the thick ascending limb of Henle. In those segments, intracellular CA2 and apical CA4 are both responsible for net transepithelial bicarbonate transport. In proximal tubules, an apical Na+/H+ exchanger, NHE3, and the vacuolar H+-ATPase secrete protons into the lumen. Through the function of CA4, these secreted H+ ions combine with luminal-filtered HCO3– to form CO2 and H2O. Then the CO2 thus formed diffuses into the cell through the apical cell membrane to form H+ and HCO3– through cytosolic CA2 enzymatic activity. The protons are recycled back into the lumen by NHE3 and the vacuolar H+-ATPase, while HCO3– diffuses passively across the basolateral membrane via a Na+-HCO3– cotransporter (29). Fish gills are well documented as being a principal organ for ion uptake and acid-base regulation mechanisms, and mitochondrion-rich cells, the major ionocytes, and/or pavement cells have been proposed as achieving these functions via the transport of H+ and/or HCO3– by exchange with Na+ and/or Cl– (9, 17, 20, 28, 35). CA has been demonstrated to play roles in ion uptake (2, 4, 32) and acid-base regulation (11, 12, 16). According to genetic databases, there are over 12 isoforms of CA reported and/or predicted in fish species including fugu and zebrafish. Whether there are specific CA isoforms responsible for acid-base regulation and ion uptake in fish gill ionocytes is still being debated. Rahim et al. (30) first purified two distinct branchial and blood CA isoforms in rainbow trout and carp, and they identified the specific existence of a branchial CA in gill ionocytes (chloride cells) and pavement cells with isoform-specific antibodies. Esbaugh et al. (8) confirmed the specific expression of the CA isoform in trout gill cells by cloning and RT-PCR analysis, and they identified the isoform as trout cytoplasmic CA, which differs from another vertebrate CA2 on the basis of phylogenetic analysis. Immunocytochemistry and Northern blot data demonstrated the expression and function of CA2 in gill ionocytes of Osorezan dace (Tribolodon hakonensis) (16). Similarly, in zebrafish embryos, CA2 was also identified in skin ionocytes by in situ hybridization with an isoform-specific probe (6, 19). On the other hand, the existence of the CA4 isoform has been reported in gill pillar cells of the spiny dogfish (Squalus acanthias) (13) and in intestinal (11, 15) and renal cells of rainbow trout (11). Apparently, so far, no data are available to demonstrate the expression and function of CA4 or its equivalent in gill ionocytes, which is the major cell type responsible for acid-base regulation and ion uptake mechanisms in freshwater fish (9, 17, 20, 28).
The purpose of the present work was to use the zebrafish (Danio rerio) as the model to test whether some CA isoforms are specifically expressed in ionocytes and whether they function in Na+ uptake and acid-base balance in fish gills. The zebrafish was selected because of its rich genetic database and applicability for various molecular physiological approaches (20). In previous studies, a novel ionocyte, H+-ATPase-rich (HR) cells, which was identified in the skin and gills of zebrafish (23), was demonstrated to be involved in acid-secretion and Na+ uptake mechanisms via H+-ATPase and NHE (6, 18, 40). Specific aims of the present study were to 1) clone and sequence the full-length or partial cDNAs of the ca gene family in zebrafish (zca); 2) identify the specific zca isoforms expressed in skin and gill ionocytes by whole mount in situ hybridization; 3) determine mRNA expression patterns of the specific zca isoforms in various tissues of zebrafish by RT-PCR; 4) determine the cellular localization of zca mRNAs, H+-ATPase, and Na+-K+-ATPase in zebrafish skin and gills; 5) elucidate the effects of translational knockdown of specific zca isoforms on H+ secretion, Na+ influx, and mRNA expressions (using real-time PCR) of the zca isoforms, zatp6v1a (H+-ATPase A-subunit) and znhe3b; and 6) determine the effects of acclimation to ambient acid or low-Na+ on the mRNA expressions of the zca isoforms, zatp6v1a and znhe3b.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Acclimation experiments.
Following a previous study (40), high-Na+ (10 meq/l) and low-Na+ (0.04 meq/l) artificial FW was prepared with double-deionized water with sufficient CaSO4·2H2O, MgSO4·7H2O, Na2SO4, NaCl, K2HPO4, and KH2PO4 added. Other ion (Ca2+, Mg2+, K+, and Cl–) concentrations and the pH of the media were the same. Local tap water (control, pH 6.7–6.9) and acidic FW (pH 4.004.05) were also prepared. 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 those in the control FW. Adult zebrafish were acclimated for 7 days to high-Na+, low-Na+, and acid FW and local tap water, and all showed normal behaviors with no mortality during the acclimation period. During the experiments, high-Na+ and low-Na+ FW was replaced every 2 days to maintain the proper ion concentrations, and acidic FW was continuously pumped into the experimental tank bottom with an electrical pump to maintain a stable pH. The pH values of all experimental media were checked with a pH meter (MP225; Mettler-Toledo, Schwerzenbach, Switzerland) and with an atomic spectrophotometer for ion concentrations (Z-5000; Hitachi, Tokyo, Japan). After 7 days of acclimation, gills were collected for the subsequent analysis.
Molecular cloning and sequences analysis. All members of the zca family were predicted from the Ensembl and NCBI databases. Subsequently, full-length or partial cDNAs of 10 zca members were cloned and sequenced from zebrafish. Amino acid sequences of ca from organisms representing different taxa were aligned and analyzed for phylogenetic and molecular evolution with MEGA software (version 4). A rooted phylogenetic tree was built using a Neighbor-joining method with bootstrap analysis for 1,000 cycles.
Preparation of total RNA. Zebrafish embryos and adult tissues were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was purified following the manufacturer's protocol. The total amount of RNA was determined by spectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE), and the RNA quality was checked by running electrophoresis in RNA-denatured gels. All RNA pellets were stored at –20°C.
RT-PCR and 5' and 3' rapid amplification of cDNA end. Total RNAs extracted from zebrafish tissues and embryos were treated with DNase I (Promega, Madison, WI) to remove genomic DNA contamination. After DNase I digestion, phenol-chloroform extraction and purification were performed to stop the reaction. For cDNA synthesis, approximately 5–10 µg of total RNA were reverse-transcribed in a final volume of 20 µl containing 0.5 mM dNTPs, 2.5 µM oligo(dT)20, 250 ng of random primers, 5 mM dithiothreitol, 40 units of an RNase inhibitor, and 200 units of SuperScript III RT (Invitrogen) for 1 h at 50°C, followed by incubation at 70°C for 15 min. For the PCR amplification, 4 µg of cDNA was used as a template in a 50-µl final reaction volume containing 0.25 mM dNTPs, 2.5 units of ExTaq polymerase (Takara, Shiga, Japan), and 0.2 µM of each primer. Thirty cycles were performed for each reaction. The primer sets for the PCR analysis of the expression patterns in different tissues are listed in Table 1. The specific primers of 5' and 3' rapid amplification of cDNA end (RACE) (Table 2) were designed from the partial sequences obtained from the cloned PCR products (data not shown). 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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Whole mount immunocytochemistry.
For triple staining of zca mRNA, Na+-K+-ATPase, and H+-ATPase, some in situ-hybridized samples were subjected to immunocytochemistry. After being washed with PBS, samples were incubated with 3% bovine serum albumin and 5% normal goat serum for 30 min to block nonspecific binding. The samples were then incubated overnight at 4°C with an 
5 monoclonal antibody against the -subunit of the avian Na+ pump (Developmental Studies Hybridoma Bank, University of Iowa, Ames, IA), and a polyclonal antibody against the A subunit of killifish H+-ATPase (22). After being rinsed with PBS for 20 min, the samples were further incubated in goat anti-rabbit IgG conjugated with FITC and goat anti-mouse IgG conjugated with Texas red (Jackson Immunoresearch Laboratories, West Grove, PA) for 2 h at room temperature. For quantification of functional HR cells, concanavalin A was used to label apical opening of HR cells following Horng et al. (18). Live embryos were preincubated in zebrafish solution containing 0.5 mg/ml Alexa Fluor 488-conjugated concanavalin A (Invitrogen) for 10 min. After being washed, embryos were imaged, and the cell numbers on the yolk sac were counted.
Surface pH of zebrafish embryos. By following previously described methods (23, 34), an anesthetized zebrafish embryo was laid laterally in the chamber for the following measurement. Briefly, microelectrodes with a tip diameter of 3–4 µm were pulled from glass capillary tubes (model TW 150-4; World Precision Instruments, Sarasota, FL) with 1.1- and 1.5-mm inner and outer diameters, respectively, then baked at 200°C overnight, and vapor-silanized with dimethyl chlorosilane (Fluka, Buchs, Switzerland) for 30 min. The microelectrodes were backfilled with a 1-cm column of 100 mM KCl/H2PO4 (pH 7.0) and then frontloaded with a 20- to 30-µm column of liquid ion exchanger cocktail (hydrogen ionophore I-cocktail B; Fluka, Seelze, Germany). The microelectrode was positioned with a stepper-motor-driven three-dimensional (3-D) positioner (Applicable Electronics, East Falmouth, MA). Data acquisition, preliminary processing, and control of the 3-D electrode positioner were performed with ASET software (Science Wares, East Falmouth, MA). The microelectrode system was attached to an Olympus upright microscope (BX-50WI) equipped with a charge-coupled device camera. The Nernstian properties of each microelectrode were measured by placing the microelectrode in a series of standard pH solutions (pH 6, 7, and 8). By plotting the voltage output of the probe against the log H+ concentration, a linear regression yielded a Nernstian slope of 57.9 (SD 2.5) (n = 5). For the inhibitor experiment, bafilomycin A1 (an inhibitor of H+-ATPase; Sigma) and amiloride (an inhibitor of NHE; Sigma) were dissolved in DMSO. Zebrafish embryos at 72 h postfertilization (hpf) were incubated in FW containing 10 µM bafilomycin A1 (23) or 100 µM amiloride (6) for 1 h and were then subjected to the measurement of surface pH as described above.
Na+ influx.
Tracer media were prepared by adding appropriate amounts of 24NaHCO3 (prepared in a 1-mW Tsing Hwa Open-Pool Reactor, Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan) to give a final working specific activity of 24Na+ of 20,000–36,000 cpm/µmol. Zebrafish embryos were transferred to the tracer medium for a 4-h incubation. After incubation, embryos were washed several times in isotope-free water medium. Twenty embryos were pooled into one vial as a sample and were analyzed for the absorbed 24Na+ with a 
-counter (B5002; Packard, Meriden, CT). The Na+ influx was calculated using the following formula according to a previously described method (5):
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Real-time polymerase chain reaction. Total RNA was extracted and reverse-transcribed from zebrafish embryos or gill tissues as described above. mRNA expressions of target genes were measured by quantitative real-time PCR (qRT-PCR) with an ABI Prism 7000 sequence analysis system (Applied Biosystems, Foster City, CA). Primers for all genes were designed (Table 4) using Primer Express software (version 2.0.0, Applied Biosystems). PCRs contained 3.2 ng of cDNA, 50 nM of each primer, and Universal SYBR green master mix (Applied Biosystems) in a final volume of 20 µl. All qRT-PCR reactions were performed as follows: 1 cycle of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min (the set annealing temperature of all primers). PCR products were subjected to melting-curve analysis, and representative samples were electrophoresed to verify that only a single product was present. Control reactions were conducted with sterile water to determine the levels of the background and genomic DNA contamination. The standard curve of each gene was confirmed to be in a linear range with β-actin as an internal control.
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Statistical analysis. Values are presented as means ± SD and were compared using Student's t-test or ANOVA (Tukey's pairwise comparison).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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mRNA expressions of zca isoforms in various tissues. For comparisons, four zca isoforms (zca2-like a, zca2-like b, zca4a, and zca15a) were selected and subjected to RT-PCR analysis in various tissues from zebrafish adults. As shown in Fig. 4, both zca2-like a and zca2-like b mRNAs were ubiquitously expressed in all tissues examined, whereas the expression patterns in zca4a and zca15a were more specific. zca4a transcripts were mainly found in the brain, eyes, and muscles and moderately in the intestines, heart, and spleen, but were undetected in other tissues, including the blood, gills, kidneys, testis, and ovaries. Notably, zca15a was the major isoform expressed in the gills and showed only a very mild amount of transcripts in the spleen, muscles, and testis (Fig. 4).
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The specificity and effectiveness of the zca MOs were confirmed by injecting embryos with zca2-like a (or zca15a):GFP cRNA (Fig. S4). After injection of zca2-like a (or zca15a):GFP cRNA, the embryos showed strong GFP expression (Fig. S4, A and C). On the other hand, coinjection with the zca2-like a (or zca15a) MO was sufficient to abolish GFP expression in the embryos (Fig. S4, B and D), indicating the specificity and effectiveness of the MOs used.
Effects of knockdown of zca isoforms on H+ activity on the embryonic surface.
A noninvasive H+-selective microelectrode was used to assay the effects of knockdown on H+ activity on the surface of zebrafish embryos. An external pH gradient (
pH) at the surface of intact embryos was measured at a location near the lower part of the yolk sac, as reported previously (18) as showing the highest H+ activity (i.e., the lowest pH). As shown in Fig. 5, injection of zca2-like a morpholinos did not initially affect the surface pH gradient in morphants at 24 hpf, but it induced a significant decline in surface H+ activities from 48 hpf and later compared with that in the wild type.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Tufts et al. (36) and Esbaugh et al. (8) pointed out the inappropriateness of directly applying mammalian CA nomenclature to teleosts. However, the phylogenetic analysis of the present study on additional teleostean CA isoforms provides additional clues to vertebrate CA's evolutionary history. The mammalian (or terrestrial) CA1/CA2/CA3/CA13 gene cluster was generated more recently after the divergence with the teleost common ancestor, and teleostean CA2-like a/CA2-like b gene duplication occurred in a whole genome duplication of the teleost common ancestor (21). To prevent confusion, the present analysis suggests annotating these isoforms as CA2-like a and CA2-like b rather than as cytoplasmic CA. In our previous studies (19, 20, 40), we annotated zCA2-like a as zCA2 and zCA15a as zCA4-like, because the nomenclatures were roughly made on the basis of BLAST (Basic Local Alignment Search Tool) similarities to known mammalian CA isoforms. These indicate the importance of the entire genome database and a functional genomic approach for identification and functional analysis of specific isoforms of ion transporters (see the discussion below).
CA has long been known to play important roles in acid-base balance and ion uptake in fish gills. So far, some studies using immunocytochemistry and in situ hybridization have demonstrated the existence of CA2-like (or cytoplasmic CA) (12) in gill mitochondrion-rich cells and/or pavement cells in rainbow trout (12, 30), flounder (Platichthys flesus) (33), mudskipper (Periophthalmodon schlosseri) (38), Osorezan dace (Tribolodon hakonensis) (16), and embryonic zebrafish (6). The present study confirmed the colocalization of zca2-like a mRNA in zebrafish skin and gill HR cells and further found an increase in zca2-like a-expressing cells following development, reflecting the enhanced acid-secretion ability of developing zebrafish (18). On the other hand, no convincing evidence for the expression of CA4 or equivalent CA isoforms in teleost gill ionocytes is available, although immunocytochemical studies indicated localization of CA4 in dogfish (Squalus acanthias) gill pillar cells (13) and cells in other osmoregulatory organs (kidneys and intestines) of rainbow trout (11, 15). The present study for the first time demonstrates the specific localization of zca15a mRNA in HR cells in the skin and gills of zebrafish. On the other hand, NHE3b (40) and NBC1 (16) were found to be colocalized in apical and basolateral gill HR cells. Taking all of these data together, the molecular evidence supports the proposed model for zebrafish HR cells, which is similar to human proximal tubular cells in the expressions of relevant transporters and enzymes (20, 40). zCA15a has a glycosylphophatidylinositol lipid anchor, through which the enzyme may also be tethered to the outer leaflet of the plasma membrane, as are the trout and human CA4 in apical and basolateral membranes of kidney cells (11, 29). However, localization of the protein of zCA15a in HR cells with a specific antibody remains to be done in the future.
Knockdown of the translations of either zca2-like a or zca15a with specific morpholinos affected the H+ concentration around the surface of the yolk sac membrane and the whole body Na+ influx, providing molecular physiological evidence to support the roles of these two CA isoforms in the mechanisms of acid-base regulation and Na+ uptake in zebrafish. The translational knockdown of zca15a showed a direct effect, an increase in the apical H+ concentration, initially at 24 hpf (23.5 h after injection of the morpholino) (Fig. 6). However, compensatory responses were subsequently observed in both zca15a and zca2-like a morphants, which showed a decrease in the apical H+ concentration by 96 hpf (Figs. 5 and 6). The cytosolic CA2-like a hydrates CO2 to form H+ and HCO3–, and the proton recycles back out of the cell by apical H+-ATPase and NHE, as proposed in mammal renal cells (29). Based on this, the decreased H+ concentration around the surface of the yolk sac membrane in both morphants at 96 hpf may be thought to have directly resulted from the translational knockdown of the two zca genes. However, one should also consider the effects derived from other related transporters and enzymes (H+-ATPase, NHE3b, NBC1, and CA15a) that have been proposed to be involved in acid secretion/Na+ uptake mechanisms of zebrafish HR cells (6, 18, 20, 23, 40). In zca2-like a morphants at 96 hpf, both upregulation of zca15a and downregulation of zatp6v1a (Fig. 8, A and B) may cause a decrease in the extracellular H+ concentration at the apical side of HR cells. The upregulation of CA4 or other membrane-bound CA isoforms has also been reported in CA2-deficient mice and is considered to be a compensatory response (3, 26). Downregulation of zatp6v1a, which deceased the apical H+ secretion from HR cells (18, 23), was also found in zca15 a morphants at 96 hpf (Fig. 8E). On the other hand, both morphants showed upregulation of znhe3b (Fig. 8, C and F). Operation of zNHE3b appears not to contribute significantly to the H+ gradient outside of the apical side of HR cells, since a supplementary experiment showed that amiloride (an inhibitor of NHE) did not decline the H+ gradient, compared with bafilomycin A1 (a H+-ATPase inhibitor) which caused 35% decrease (Fig. S5). It should be noted that the decline of H+ gradient in zca2-like a and zca15a morphants was approximately 20–36% (Figs. 5 and 6).
The decline in the extracellular H+ concentration at the apical side of HR cells reasonably favors the operation of NHE3b, which can be driven by the H+ gradient (25). The present data, presenting increased Na+ influx (Fig. 7A) and upregulation of znhe3b (Fig. 8C), support this notion. Congenital CA2 deficiency in mammals has reported to cause renal tubular acidosis (3). zca2-like a knockdown may induce a systemic acidosis, which may also contribute to the favorable gradient for the operation of zNHE3b in HR cells, but this needs further confirmation. In a recent work, Yan et al. (40) examined the mRNA expressions of gill zatp6v1a and znhe3b in zebrafish acclimated to a low-Na+ environment, and indicated that apical H+-ATPase was downregulated but probably maintained an intracellular H+ gradient to facilitate Na+ uptake via apical NHE3b, which may be the dominant player, and thus its function was enhanced. The present study supports this hypothesis by providing further molecular physiological data. In both zca morphants, downregulated zatp6v1a and upregulated znhe3b were accompanied by an increase in Na+ uptake and a decline in the apical proton concentration. All these demonstrate the partitioning and negative correlation of NHE3b and H+-ATPase for involvement in the Na+ uptake/acid-base regulation mechanisms in zebrafish HR cells.
Subsequent acclimation experiments further supported the roles of zCA2-like a and zCA15a in Na+ uptake/acid-base regulation mechanisms in zebrafish HR cells. After 1-wk acclimation to low-pH FW, fish had to compensate for internal acidosis by enhancing the proton secretion and HCO3– uptake, which is apparently achieved by stimulating H+-ATPase (40) and CA15a (the present study), respectively, in zebrafish HR cells. Stimulation of the expression and function of gill H+-ATPase in hypercapnia- or acid-acclimated fish has been reported in many previous studies (10, 14, 27, 28, 40). Enhancing CA4 expression to facilitate HCO3– uptake (via subsequent operations of cytosolic CA2 and basolateral NBC) as compensation for acidosis has been reported in fish kidneys (11), and the present study for the first time demonstrates a similar mechanism in fish gills. Interestingly, acclimation to low-Na+. FW also induced stimulation of zca15a expression (Fig. 9B). Apparently, the mechanism of apical CA4 facilitating the operation of NHE3 in mammal proximal tubular cells (29) also holds in zebrafish gill HR cells. Enhancement of zca15a expression in zebrafish HR cells decreases the apical proton concentration (as occurred in zca2-like a morphants at 96 hpf), providing a more favorable gradient for the operation of zNHE3b to absorb more Na+ for compensation.
Interestingly, zca15a morphants showed no significant change in the expression of zca2-like a (Fig. 8D). CA2 mRNA expression also did not change in mice with genetic knockout of CA9 (26), which is a membrane-associated CA isoform like CA4 (29). On the other hand, zca2-like a expression was not changed in either the acid or low-Na+ environment (Fig. 9A). It is probable that the intact CA2-like a is sufficient to overcome the physiological defects caused by the translational knockdown of zca15a, and it may provide sufficient carbonic anhydrase activity to fulfill the physiological needs in zebrafish coping with different environments. Indeed, in mammalian kidneys, CA2 accounts for >95% of CA activity and shows the highest catalytic rate, whereas CA4 and other membrane-associated isoforms sustain the remaining 5% of activity (3, 29, 39).
In summary, the present study provides molecular physiological evidence for the existence of zCA2-like a and zCA15a in zebrafish HR cells and for the roles of the two CA isoforms in Na+ uptake/acid-base regulation mechanisms. Among the 20 isoforms identified in the present study, only 10 of them have been cloned and examined for mRNA localization. Whether any other(s) in the remaining 10 members is expressed in gill ionocytes and involved in ion regulation mechanisms remains to be studied in future.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* Z.-Y. Lin and B.-K. Liao contributed equally to this work. 
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