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Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 Contents lists available at SciVerse ScienceDirect Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa Effects of salinity acclimation on Na +/K +–ATPase responses and FXYD11 expression in the gills and kidneys of the Japanese eel (Anguilla japonica) Cheng-Hao Tang a, b, Dong-Yang Lai c, Tsung-Han Lee c, d,⁎ a Institute of Marine Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan d Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan b c a r t i c l e i n f o Article history: Received 8 May 2012 Received in revised form 25 July 2012 Accepted 30 July 2012 Available online 3 August 2012 Keywords: Na+/K+–ATPase FXYD protein Cortisol Japanese eel a b s t r a c t Na +/K +–ATPase (NKA) is a primary active pump provides the driving force for ion-transporting systems in the osmoregulatory tissues of teleosts. Therefore, modulation of NKA expression or activity and its regulatory subunit, FXYD protein, is essential for teleosts in salinity adaptation. To understand the mechanisms for modulation of NKA in catadromous fishes, NKA expression and activity, cloning and mRNA expression of FXYD11 (AjFXYD11) were examined in Japanese eel (Anguilla japonica) exposed to fresh water (FW) and seawater (SW; 35‰). Expression and activity of NKA as well as mRNA expression of AjFXYD11 in gills were elevated in SW eel compared to FW eel. Conversely, NKA responses in eel kidneys were higher in FW group than SW group, whereas no significant difference was found in renal AjFXYD11 expression between the two groups. Comparison of NKA activity and AjFXYD11 expression between two osmoregulatory tissues suggested that AjFXYD11 plays a specific, functional role in gills. However, since cortisol plays an important role for regulation of ion transport in teleost SW acclimation and gill AjFXYD11 expression was elevated in SW eel, the organ culture approach was used to study the effect of cortisol on gill AjFXYD11 mRNA expression. Our results revealed that cortisol treatment increased the levels of gill AjFXYD11 transcripts. This finding suggested that cortisol could be involved in the regulation of NKA by altering AjFXYD11 expression during the process of SW acclimation in A. japonica. Taken together, the differential expression of branchial and renal NKA and AjFXYD11 implicated their roles in the osmotic homeostasis of Japanese eel exposed to environments of different salinities. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Maintaining a stable internal environment is essential for animals to adapt to a variety of habitats. In response to changes in environmental salinity, ion-transporting epithelia play crucial homeostatic roles in modulating ion fluxes. Moreover, gills and kidneys are the most important organs for osmoregulation in teleosts (Marshall and Grosell, 2006). The fundamental transporters responsible for ion movement across gill and kidney epithelia have been widely studied (Perry et al., 2003; Evans et al., 2005; Marshall and Grosell, 2006; Katoh et al., 2008; Hwang et al., 2011). Among these transporters, Na +/K +–ATPase (NKA) is a membrane-spanning protein that couples the exchange of two extracellular K + ions for three intracellular Na + ions to the hydrolysis of one molecule of ATP (Post and Jolly, 1957). Because NKA is a primary active pump (Blanco and Mercer, 1998; Bystriansky and Kaplan, 2007), it is crucial not only for sustaining ⁎ Corresponding author at: Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan. Tel.: +886 4 2285 6141; fax: +886 4 2285 1797. E-mail address: [email protected] (T.-H. Lee). 1095-6433/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2012.07.017 intracellular homeostasis, but also for providing a driving force for many transporting systems within fish gills and kidneys (Hirose et al., 2003; Perry et al., 2003; Marshall and Grosell, 2006; Hwang and Lee, 2007). Changes in NKA activity occur in the osmoregulatory organs of animals when confronted with changes in environmental salinity (Marshall, 2002; Hirose et al., 2003; Burg et al., 2007). Moreover, NKA activity is tightly regulated by various factors including intracellular Na+ concentration, changes in gene and protein expression of its essential subunits, isoform switching and phosphorylation (Blanco and Mercer, 1998; Therien and Blostein, 2000; Feraille and Doucet, 2001; Richards et al., 2003; Bystriansky and Kaplan, 2007). In addition, small regulatory subunits of NKA including several members of the FXYD protein family have been identified that may play an important role in modulating NKA activity. The transmembrane domain and the FXYD motif of FXYD proteins have been shown to bind to the α- and β-subunits of NKA and modulate NKA activity by affecting the apparent affinities for intracellular Na+ and extracellular K+ ions (Pu et al., 2001; Crambert et al., 2002; Garty et al., 2002; Geering et al., 2003; Crambert et al., 2005; Garty and Karlish, 2006; Geering, 2006; Delprat et al., 2007). It is well known that modulation of NKA activity is important for acclimation of fish to C.-H. Tang et al. / Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 hypotonic and hypertonic environments (Marshall, 2002; Hwang and Lee, 2007). Although the potential role of FXYD proteins in fish has received less attention than in mammals, some orthologous and paralogous FXYD members have been studied in the elasmobranch, spiny dogfish (Squalus acanthias) (Mahmmoud et al., 2000, 2003), freshwater (FW) teleost, zebrafish (Danio rerio) (Saito et al., 2010), anadromous teleost, Atlantic salmon (Salmo salar) (Tipsmark, 2008; Tipsmark et al., 2010) and euryhaline species, including the spotted green pufferfish (Tetraodon nigroviridis) (Wang et al., 2008), and tilapia (Oreochromis mossambicus) (Tipsmark et al., 2011). Nevertheless, little is known about the role of FXYD proteins in the osmoregulatory organs of catadromous teleost. Japanese eel (Anguilla japonica) is a catadromous species with remarkable euryhalinity (Kaneko et al., 2008). To survive in a wide range of environmental salinities throughout their life cycle or different cultured conditions, the eel is equipped with mechanisms for acclimation to both hypotonic and hypertonic environments. Moreover, increase in branchial NKA activity was observed in eel adapted to hypertonic condition (Marshall, 2002; Hirose et al., 2003; Hwang and Lee, 2007). Recent studies indicated that FXYD11 mRNA is highly expressed in fish gills and its expression levels are altered by changes of environmental salinities (Saito et al., 2010; Tipsmark et al., 2010; 2011). It is therefore hypothesized that FXYD11 may be involved in the regulation of NKA activity in the catadromous eel. To test this hypothesis and clarify the gill specificity of FXYD11 in the catadromous teleost, we investigated the correlation between FXYD11 expression and NKA expression (i.e., mRNA and protein expression and specific activity) in osmoregulatory organs (gill and kidney) of A. japonica acclimated to fresh water (FW) and seawater (SW). In addition, because the corticosteroid, cortisol, is an important hormone involved in hypo- and hyperosmoregulation (Sakamoto et al., 2001; Marshall and Grosell, 2006; Sakamoto and McCormick, 2006), we used an in vitro organ culture to evaluate whether cortisol plays a role in the osmoregulation of eel gills by affecting the expression levels of AjFXYD11. This study assessed the effects of environmental salinity and cortisol signaling on FXYD11 expression to explore the possible role of FXYD11 in salinity adaptation in a catadromous species. 2. Materials and methods 2.1. Fish and experimental environments Yellow Japanese eel (A. japonica) with a body mass of 200±14.5 g and total length of 53.4±2.2 cm were obtained from a fish farm in Taiwan. SW (35‰) was prepared from local tap water supplemented with the appropriate amounts of synthetic sea salt (Aquarium Systems, Mentor, OH, USA). Eels were reared in FW or SW at 21±1 °C with a daily 12 h photoperiod for at least one week before experiments. Feeding was terminated 48 h prior to the following experiments. The water was continuously circulated through fabric-floss filters, and fish were fed commercial pellets daily. The use protocol for the experimental fish was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Chung Hsing University (IACUC Approval No. 98–103) to T.-H.L. 2.2. Tissue collection The eels were anesthetized by immersion in 100 mg/L MS-222 diluted in aquarium water (Sigma-Aldrich, St Louis, MO, USA). Following anesthesia, the fish were decapitated and sampled immediately after sacrifice. The studied tissues (i.e., gills, kidneys, intestine, liver, muscle and skin) were removed. For RNA extraction and immunoblotting analysis, the organs were directly immersed into liquid nitrogen immediately after removal and subsequently homogenized. 303 2.3. Cloning of fxyd11 in A. japonica Using the European eel (Anguilla anguilla) transcriptome database (EeelBase; http://compgen.bio.unipd.it/eeelbase/), we retrieved AaFXYD11 (name: eu_c14786) gene sequences. To clone A. japonica FXYD11 (AjFXYD11), the AaFXYD11 sequence was used as a template to design the primer pair for conducting reverse transcription polymerase chain reaction (RT-PCR). The primer pair for AjFXYD11 and glyceraldehyde-3-phosphate dehydrogenase (AjGAPDH; Accession number: AB075021) (as an internal control) were designed using Primer3 software (http://frodo.wi.mit.edu/primer3/input.htm). The sequences of primer pairs for AjFXYD11 and AjGAPDH were as follows (5′ to 3′): AjFXYD11, forward: TTGAGCTGCTAGAAGATG and reverse: GTCAATCTATGGCTCCTC; AjGAPDH, forward: GCGCCAGCCAGA ACATCATC and reverse: CGTTAAGCTCGGGGATGACC. 2.4. Total RNA extraction and RT-PCR analysis Each total RNA sample from the various tissues of eel was extracted with the TriPure Isolation Reagent (Roche, Mannheim, Germany) following the manufacturer's instructions. The RNA pellet was dissolved in 30 μL of sterilized DDW (i.e., distilled and deionized water) and treated with the RNA clean-up protocol from the RNAspin Mini RNA isolation kit (GE Health Care, Piscataway, NJ, USA) following the manufacturer's instructions, to eliminate genomic DNA contamination. Extracted RNA samples were stored at −80 °C after isolation. The concentration and quality of extracted RNA were measured by NanoDrop 2000 (Thermo, Wilmington, DE, USA). Purified RNA with an A260/A280 ratio between 1.8 and 2.0 was used for all RNA experiments. First-strand cDNA was synthesized from 2 μg of total RNA by iScript™ cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions. For RT-PCR analysis, 2 μL of first-strand cDNA was used as a template in a 50 μL final reaction volume containing 0.2 mM dNTP, 1.25 units of Ex Taq polymerase (Takara, Otsu, Shiga, Japan), and 0.2 μM of each primer. The reaction of PCR amplification of AjFXYD11and AjGAPDH was performed under the following conditions: 5 min denaturation at 95 °C for one cycle followed by 29 cycles of denaturation at 95 °C (1 min), annealing at 56 °C (1 min) for AjFXYD11or 60 °C (1 min) for AjGAPDH, extension at 72 °C (30 s), and a final single extension at 72 °C for 5 min. The PCR products were analyzed by 1.5% agarose gels containing the Safeview DNA stain (GeneMark, Taipei, Taiwan) to confirm the expected molecular weight of a single amplification product. The nucleotide sequence of the PCR products was examined by direct sequence analysis (Tri-I Biotech, Taipei, Taiwan). 2.5. Real-Time PCR Expression levels of AjFXYD11 and Na +/K+–ATPase (AjNKA) α-subunit (ATP1A1) (Accession number: X76108) (Cutler et al., 1995; Tse et al., 2006) were quantified by MiniOpticon real-time PCR system (Bio-Rad Laboratories). Real-time PCR primers used to analyze gene expression of AjFXYD11and NKA α-subunit were designed using Primer3 software (http://frodo.wi.mit.edu/primer3/input.htm) with the following sequences (5′ to 3′): AjFXYD11, forward: CTTATCCTCAGCATCACC CG and reverse: GGCTACGAATTGGAGGCTTG; AjNKA α-subunit, forward: TCTGATGTCTCCAAGCAGGC and reverse: CTGGTCAGGGTGTAG GC; AjGAPDH, forward: GCGCCAGCCAGAACATCATC and reverse: CGTT AAGCTCGGGGATGACC. A standard curve was constructed for each target gene to optimize the template cDNA concentration and to verify that the threshold cycle (Ct) fell into an acceptable range. The amplification efficiency for standard curves of the primer pairs used for real-time PCR analysis was within the range of 95–100%. Real-time PCR reactions contained 8 μL of cDNA (20× dilution), 0.5 μM of primer pair, and 10 μL of 2× SYBR Green Supermix (Bio-Rad). The conditions for all real-time PCR reactions were as follows: 95 °C for 5 min, followed by 40 cycles of 98 °C for 10 s and 60 °C for 20 s. All samples were run in triplicate. 304 C.-H. Tang et al. / Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 Non-template control reactions were conducted with sterile deionized water instead of cDNA template to determine the background signal levels. A melting curve analysis was performed after each reaction to confirm the efficiency of the conditions for real-time PCR and to verify that no primer dimers or other nonspecific products were synthesized during the reactions. AjGAPDH was used as an internal control for normalization of AjFXYD11 and AjNKA α-subunit since the mRNA expression of AjGAPDH was similar in different tissues and with salinity and cortisol treatments in our preliminary test. For each unknown sample, the comparative Ct method with the following formula: 2 ^ [(Cttarget, n − CtGAPDH, n) − (Cttarget, c − CtGAPDH, c)] (Livak and Schmittgen, 2001) was used to obtain the corresponding values of target gene where Ct corresponded to the threshold cycle number. and incubated at room temperature (26–28 °C) for 2 h with secondary antibody (1:12,000 dilution). The immunoreactive bands were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and imaged with a ChemiDoc (Bio-Rad). To verify even loading of the crude membrane fractions, the total protein amount of each lane on the blots was quantified after staining the membranes with Ponceau S (Romero-Calvo et al., 2010) and analyzing images of the blot using MCID software (ver. 7.0, rev. 1.0; Imaging Research, St. Catharines, ON, Canada). Results were converted to numerical values to compare the relative protein abundance of the immunoreactive bands. The relative protein abundance of branchial and renal NKA α-subunit protein in each sample was defined as the ratio of antibody/Ponceau S. 2.6. Preparation of crude membrane fractions 2.9. Specific NKA activity The procedure of preparation of crude membrane fractions of gill and kidney was performed according to Tang and Lee (2011). The eel were anesthetized by immersion in 100 mg/L MS-222 diluted in aquarium water (Sigma) and then the gills and kidneys of the fish were excised and blotted dry immediately. The samples were immersed in liquid nitrogen and placed into ice-cold homogenization buffer (250 mM sucrose, 1 mM EDTA, 30 mM Tris, pH 7.4). Homogenization was performed in 2 mL tubes using the Polytron PT1200E homogenizer (Lucerne, Switzerland) at maximal speed for 15 s. Debris, nuclei, and lysosomes were removed by low-speed centrifugation (13,000 g for 10 min, 4 °C). The remaining supernatant was centrifuged at medium speed (20,800 ×g for 1 h, 4 °C). The resulting pellet was resuspended in homogenization buffer and stored at −80 °C. The pelleted fraction contained large fragments of the plasma membrane along with membranes from the Golgi and the endoplasmic reticulum, but no small cytoplasmic vesicles as they typically do not pellet down unless greater forces (100,000×g for> 1 h) are applied (Alberts et al., 1994). This fraction is therefore referred to as the crude membrane fraction. Aliquots of crude cell membrane fractions were saved for protein determination analysis. Protein concentrations were determined with BCA Protein Assay Kit (Pierce, Hercules, CA, USA) using bovine serum albumin (Pierce) as a standard. The crude membrane fractions were stored at −80 °C until the immunoblotting experiments. A method using 96-well microplate to measure the inorganic phosphate concentrations for determination of NKA activity was performed according to Tang et al. (2010) with minor modification. Aliquots of the suspension of the gill and kidney crude membrane fractions, prepared as described above, were used to determine the protein concentration and NKA enzyme activity. The reaction medium (final concentration: 100 mM imidazole–HCl buffer, pH 7.6, 125 mM NaCl, 75 mM KCl, 7.5 mM MgCl2) was prepared according to Hwang et al. (1988). Then 10 μL crude membrane fractions, 50 μL 10 mM ouabain (specific inhibitor of NKA) or deionized water and 100 μL 10 mM Na2ATP were added to 340 μL of the reaction medium. The enzyme activity was defined as the difference between the inorganic phosphate liberated in the presence and absence of ouabain in the reaction mixture. The reaction mixture was incubated at 28 °C for 20 min followed by immediate ice bath for 10 min to stop the reaction (Cheng et al., 1999). Because van der Heijden et al. (1997) and Sardella et al. (2008) have demonstrated that the specific NKA activity was measured at the exposed temperature of fish would correlate with the level of in vivo activity. Therefore, the reaction was run at 28 °C in this study. The concentration of inorganic phosphate was measured according to Doulgerakia et al. (2002). The colorimetric reagent consisted of 1% Tween-20 and 0.75% ammonium molybdate in 0.9 M H2SO4. The reaction mixtures and colorimetric reagent were mixed in a 1:1 (v/v) ratio and then the concentration of inorganic phosphate in each of the samples was determined by a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA, USA) at 405 nm. Each sample was determined in triplicate. Some protocols determine the concentration of inorganic phosphate by measuring the color of molybdenum blue, which is the reduced product of phosphomolybdate. The instability of color formation and reagents, however, were variables in those protocols. The formation of the unreduced phosphomolybdate in the present study is directly proportional to the amounts of inorganic phosphate. 2.7. Antibodies The primary antibody used in this study was α5, a mouse monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) raised against the α-subunit of avian NKA. The secondary antibody used in this study was horseradish peroxidase (HRP)-conjugated goat antimouse IgG (Jackson Immunoresearch, West Baltimore Pike, PA, USA). 2.8. Immunoblotting Immunoblotting procedures were performed according to Tang and Lee (2011). Proteins of the membrane fraction were heated together with the 6 × sample buffer (12% SDS and 30% glycerol, 62.5 mM Tris (pH 6.8), 0.6 M dithiothreitol, 0.06% bromophenol blue) at 37 °C for 30 min. The prestained protein molecular weight marker was purchased from Fermentas (SM0671; Hanover, MD, USA). All samples were separated by electrophoresis on sodium dodecyl sulfate-containing 7.5% polyacrylamide gels (15 μg of protein/lane). The separated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) by electroblotting. After preincubation for 3 h in PBST buffer (phosphate-buffered saline containing 0.075% Tween-20) containing 5% (w/v) nonfat dried milk to minimize nonspecific binding, the membranes were incubated for 2 h at room temperature (26°–28 °C) with primary antibody (α5) diluted in 1% bovine serum albumin (BSA) and 0.05% sodium azide in PBST (1:5000 dilution). Then, the membranes were washed in PBST 2.10. Organ culture The effect of a single dose of cortisol (10 μg/mL) on the mRNA expression level of branchial AjFXYD11 was examined in vitro using the organ culture approach. Preparation of gill filaments for organ culture was performed following the methods previously described in McCormick and Bern (1989). The culture medium was Leibovitz-15 (L-l 5) containing 292 μg/mL L-glutamine with 25 mM N-2-hydroxyethylpiperazine-N′-2ethanesulfonic acid (HEPES) buffer and 4 mg/mL BSA (GeneMark) was added immediately before use and adjusted to pH 7.5 with NaOH. Gill arches from FW-acclimated eel were excised and placed in sterile Petri dishes containing sterile L-l5 placed on ice. Gill filaments were separated immediately above the septum and gently dissociated by passage through a pipette. Excised gill filaments were placed in 2 mL L-15 containing 500 U/mL penicillin and 500 μg/mL streptomycin. Gill filaments were preincubated in this media at 20–22 °C for 4 h with gentle shaking. C.-H. Tang et al. / Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 Then gill filaments were transferred to L-15 containing 50 U/mL penicillin and 50 μg/mL streptomycin with or without 10 μg/mL cortisol sodium-hydrocortisone-hemisuccinate (Sigma). The dosage of cortisol has been used in previous studies (Kiilerich et al., 2007; Tipsmark et al., 2010). After 18 h, the incubation was terminated by freezing the gill blocks in liquid nitrogen (Tipsmark et al., 2010). Total RNA extraction and real-time PCR analysis were executed as described above. 2.11. Analysis of plasma osmolality and muscle water contents (MWC) A. japonica blood was collected from the heart with 1 mL heparinized syringes and 29 G needles, and then the blood samples were centrifuged at 1000 ×g at 4 °C for 10 min. Plasma osmolality was measured immediately by using the Wescor 5520 Vapro Osmometer (Logan, UT, USA). The procedure used to measure MWC was conducted according to Tang et al. (2009). MWC was measured gravimetrically after drying the samples at 100 °C for 48 h. 2.12. Statistical analysis In the acclimation experiments, statistical significance was determined using Student's t-test (P b 0.05) for group data analysis. Values were expressed as means ± S.E.M. 305 3. Results 3.1. mRNA and protein expression and activity of Na +/K +–ATPase (NKA) in gills and kidneys Real-time polymerase chain reaction (PCR) analysis revealed that the relative mRNA levels of gill NKA α-subunit were significantly higher in SW-acclimated Japanese eel (A. japonica) than fresh water (FW)-acclimated individuals (Fig. 1A). Quantification of single, 105 kDa immunoreactive bands (Fig. 1B) from each group showed that the relative protein abundance of the SW-acclimated individuals were significantly higher than FW-acclimated individuals (Fig. 1C). Furthermore, the pattern of branchial NKA specific activity was positively correlated with branchial mRNA and protein levels and activity of gill NKA was significantly elevated in SW group compared with FW group (Fig. 1D). The pattern of renal NKA mRNA and protein expression and specific activity was different from the pattern observed in gills. The transcript levels of renal NKA α-subunit were higher in FW eel than SW eel (Fig.2A). Immunoblotting analysis showed that renal NKA α-subunit in FW and SW eel also resulted in a single immunoreactive band approximately 105 kDa (Fig. 2B). However, the protein abundance of renal NKA α-subunit in the FW-acclimated eel was significantly higher than that in SW-acclimated eel (Fig. 2C). Similarly, NKA specific activity in kidneys of FW group was higher than that in SW group (Fig. 2D). Fig. 1. Branchial Na+/K+–ATPase (NKA) expression and activity in Japanese eel (Anguilla japonica) acclimated to fresh water (FW) and seawater (SW); n = 5 for each experiment. (A) mRNA expression of gill NKA α-subunit were analyzed by real-time PCR. The expression levels were significantly higher in the SW group than the FW group. (B) A representative immunoblot of gills from eel acclimated to FW and SW probed with a primary antibody to NKA α-subunit (α5) confirming a single immunoreactive band with molecular weight of about 105 kDa. (C) Relative protein abundance of gill NKA α-subunit was significantly higher in SW eel than FW eel. (D) Gill NKA activity was measured in FW and SW acclimated eel. Gill NKA activity of SW group was significantly higher than that of FW group. The asterisk indicated a significant difference by Student's t-test (P b 0.05). Values are means ± S.E.M. M, marker. 306 C.-H. Tang et al. / Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 3.2. Japanese eel (A. japonica) FXYD11 (AjFXYD11) Bioinformatics tools including the European eel (A. anguilla) transcriptome database (EeelBase; http://compgen.bio.unipd.it/eeelbase/), the National Center for Biotechnology Information (NCBI) database, and BLAST were used to clone the AjFXYD11 gene. The entire cDNA nucleotide sequence of the AjFXYD11 was cloned with a PCR based method. The 213 bp cDNA sequence of AjFXYD11 (Accession number: JQ867188) encoded a deduced protein with 70 amino acid residues. The amino acid sequence shared 76% and 69% homology with previously published FXYD11 sequences from Atlantic salmon (S. salar) (NP_001117201 and NP_001117200) (Tipsmark, 2008) and zebrafish (D. rerio) (XP_003200301) (Saito et al., 2010), respectively. According to the hydropathy plot analysis, AjFXYD11 contained one transmembrane domain (32–54 residue of AjFXYD11 amino acid sequence; gray background in Fig. 3), which was highly conserved in the other FXYD proteins (40–70% identity). Thr60 (circled in Fig. 3) is a predicted site of phosphorylation by protein kinase C. Asn66 and Asn69 are the predicted sites of glycosylation (dotted box in Fig. 3). Based on the alignment, AjFXYD11 protein contains a conserved FXYD motif (solid line box in Fig. 3). 3.3. Tissue distribution of AjFXYD11 Reverse transcription (RT)-PCR with cDNA from the studied tissues (gills, kidneys, intestine, liver, muscle and skin) of FW eel was Fig. 3. Nucleotide sequence and deduced amino acid sequence of Japanese eel (Anguilla japonica) FXYD11 (AjFXYD11). The boxes indicate the highly similar FXYD motif (solid line) and two glycine residues (dashed line). The transmembrane domain is shaded gray. performed to determine the distribution of AjFXYD11 mRNA and glyceraldehyde-3-phosphate dehydrogenase (AjGAPDH) mRNA was used as the loading control. After 40 cycles of RT-PCR reaction with the specific primers of AjFXYD11, the expected 319 bp product was detected in gill tissue of FW eel (Fig. 4A). A more sensitive approach, real-time PCR, was further performed to quantify the mRNA abundance in the various eel tissues. AjFXYD11 mRNA expression was the strongest in gills, where the AjFXYD11 mRNA abundance was 177-fold higher than in kidneys and more than 2000-fold higher than in any other tissues. The relative order of the levels of AjFXYD11 mRNA expression in the studied Fig. 2. Renal Na+/K+–ATPase (NKA) expression and activity in Japanese eel (Anguilla japonica) acclimated to fresh water (FW) and seawater (SW); n = 5 for each experiment. (A) mRNA expression of renal NKA α-subunit were analyzed by real-time PCR. The expression levels were significantly higher in the FW group than the SW group. (B) A representative immunoblot of kidneys from eel acclimated to FW and SW probed with a primary antibody to NKA α-subunit (α5). A single immunoreactive band with molecular weight of about 105 kDa was shown. The immunoreactive bands of FW eel were more intense than SW individuals. (C) Relative protein abundance of kidney NKA α-subunit was significantly higher in FW eel than SW eel. (D) Renal NKA activity was measured in FW and SW acclimated eel. Renal NKA activity of SW group was significantly higher than that of FW group. The asterisk indicated a significant difference by Student's t-test (P b 0.05). Values are means ± S.E.M. M, marker. C.-H. Tang et al. / Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 307 tissues was as follows: gills>kidneys>intestine>liver>muscle>skin (Fig. 4B). 3.4. Effects of environmental salinity and cortisol on AjFXYD11 mRNA expression The relative expression of AjFXYD11 mRNA in gills and kidneys of eel acclimated to FW and SW was examined by real-time PCR. Compared to FW eel, SW eel expressed higher levels of branchial AjFXYD11 mRNA (Fig. 5A). However, the levels of renal AjFXYD11 mRNA were similar between FW and SW groups (Fig. 5B). The expression of gill AjFXYD11 transcripts was higher in cortisol treatment (10 μg/mL) than control after in vitro culture for 18 h (Fig. 6). 3.5. Plasma osmolality and muscle water contents (MWC) Plasma osmolality of A. japonica acclimated to environments of different salinities was maintained within a range appropriate for physiological homeostasis, although the plasma osmolality was slightly but significantly higher in SW group (Fig. 7A). There was no significant difference in the MWC between eel exposed to FW and SW (Fig. 7B). 4. Discussion The maintenance of a stable internal environment is crucial for animals to survive in a variety of habitats. Salinity is one of the major abiotic factors that influence physiological homeostasis in aquatic organisms (Kültz and Burg, 1998; Boeuf and Payan, 2001; Deane and Woo, 2004; 2009). Ion-transporting epithelia, such as those found in gills and kidneys, play important roles in modulating ion fluxes in response to changes in environmental salinity. Among the transporters that modulate ion fluxes, Na+/K+–ATPase (NKA) is a primary active pump responsible for the active transport of Na+ out of and K+ into animal cells (Post and Jolly, 1957). NKA is essential for maintaining intracellular homeostasis as Fig. 5. mRNA abundance of gill and kidney FXYD11 in fresh water (FW) and seawater (SW) Japanese eel (Anguilla japonica) (AjFXYD11); n = 5 for each experiment. (A) mRNA levels of branchial AjFXYD11 was significantly higher in the SW group than the FW group. (B) No significant difference was found in renal AjFXYD11 expression between the FW and SW groups. The asterisk indicated a significant difference by Student's t-test (P b 0.05). Values are means ± S.E.M. well as for energizing ion-transporting systems in animals (Blanco and Mercer, 1998; Bystriansky and Kaplan, 2007; Vagin et al., 2007). In the present study, real-time PCR and immunoblotting analyses revealed that the levels of mRNA and protein expression of the NKA α-subunit were significantly higher in gills of the Japanese eel (A. japonica) acclimated to seawater (SW) than in A. japonica acclimated to fresh water (FW) (Fig. 1A–C). This suggests that the elevation of branchial NKA activity in SW A. japonica (Fig. 1D) results from Fig. 4. (A) Distribution of Japanese eel (Anguilla japonica) FXYD11 gene in freshwater (FW) A. japonica (AjFXYD11) tissues by reverse transcription (RT)-PCR analysis. A. japonica glyceraldehyde-3-phosphate dehydrogenase (AjGAPDH) was applied as an internal control for all cDNAs used in the analysis. 319 bp product was detectable in gills by RT-PCR analysis. (B) Quantitative mRNA expression of AjFXYD11 was analyzed by real-time PCR (n=5). AjFXYD11 was detected in all examined tissues and the relative expression levels among the tissues were as follows: gills>kidneys>intestines≈liver≈muscle≈skin. G, gill; K, kidney; I, intestine; L, liver; M, muscle, Sk, skin. Fig. 6. Effects of cortisol on FXYD11 mRNA expression in gill blocks from fresh water (FW) acclimated Japanese eel (Anguilla japonica) (AjFXYD11); n= 5 for all groups. mRNA abundance of gill AjFXYD11 was significantly induced by cortisol treatment. The asterisk indicated a significant difference by Student's t-test (P b 0.05). Values are means ± S.E.M. 308 C.-H. Tang et al. / Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 Fig. 7. (A) Plasma osmolality and (B) muscle water contents (MWC) in Japanese eel (Anguilla japonica) acclimated to fresh water (FW) and seawater (SW); n=5 for all groups. Plasma osmolality was significantly higher in SW-exposed (288.8±1.97 mOsm/kg) fish than FW-exposed (271.2±1.74 mOsm/kg) individuals. There was no significant difference in MWC between FW- and SW-exposed eel. increasing the transcript and protein levels of branchial NKA α-subunit. This is similar to previous reports of NKA expression or activity in tilapia (O. mossambicus) (Lee et al., 2003), pufferfish (T. nigroviridis) (Lin et al., 2004), sailfin molly (Poecilia latipinna) (Yang et al., 2009) and salmonid species (Hwang and Lee, 2007). Recently, salinity-dependent expression of two NKA α-subunit isoforms, α1a and α1b, was reported in salmonid fish (Richards et al., 2003; Bystriansky et al., 2006; 2007). It is worth to identify and analyze the expression of NKA α1a and α1b in eel to clarify the involvement of switch of two isoforms in modulation of NKA activity in eel gills. Unlike NKA expression and activity in gills, those of eel kidney are poorly understood. Our results revealed that renal NKA expression (mRNA and protein) and activity of FW-acclimated eel were higher than those of SW-acclimated eel (Fig. 2). Similarly, a significant increase in renal NKA activity has been previously reported in kidneys of other FW-acclimated euryhaline species such as killifish (Fundulus heteroclitus) (Epstein et al., 1969), thicklip gray mullet (Crenimugil labrosus) (Lasserre, 1971; Gallis and Bourdichon, 1976), European sea bass (Dicentrarchus labrax) (Lasserre, 1971; Venturini et al., 1992; Nebel et al., 2005), thinlip mullet (Liza ramada) (Gallis and Bourdichon, 1976), striped bass (Morone saxatilis) (Madsen et al., 1994), black seabream (Mylio macrocephalus) (Kelly et al., 1999), pufferfish (T. nigroviridis) (Lin et al., 2004), three sturgeon species (Acipenser ruthenus, A. gueldenstaedtii, and A. stellatus) (Krayushkina et al., 2006), and milkfish (Chanos chanos) (Tang et al., 2010). The primary function of teleost kidneys is to excrete excess water while reabsorbing most of the filtered solutes. To minimize salt loss, FW teleosts reabsorb approximately 95% of the NaCl that enters the glomerular filtrate (Miyazaki et al., 2002; Marshall and Grosell, 2006). Elevation of NKA expression and activity in FW eel kidneys might indicate the role of renal NKA in energizing ion reabsorption for the homeostasis of euryhaline teleosts exposed to hypotonic environment (Tang et al., 2010). In addition to changes in the expression of the NKA α-subunit, different members (FXYD1–7) of the FXYD protein family have been identified as regulators of NKA. For instance, FXYD protein-mediated regulation of NKA activity has been widely examined in mammals (Geering et al., 2003; Crambert et al., 2005; Garty and Karlish, 2006; Geering, 2006; Delprat et al., 2007). The first fish FXYD protein (FXYD10; Phospholemman-like) was identified in shark rectal glands (Mahmmoud et al., 2000). Recently, eight FXYD proteins (FXYD2, FXYD5–9 and FXYD11–12) have been identified in the Atlantic salmon (S. salar) and have been characterized for their tissue-specific expression and their relationship to salinity adaptation (Tipsmark, 2008). Of these eight FXYD proteins, FXYD11 has been reported to be highly expressed in fish gills (Tipsmark, 2008; Saito et al., 2010; Tipsmark et al., 2010; Tipsmark et al., 2011). In the present study, we cloned the FXYD11 gene from the catadromous teleost, Japanese eel (AjFXYD11), which we identified based on homology with the characteristic FXYD motif and the possession of a transmembrane domain similar to other vertebrate FXYD proteins (Fig. 3). The FXYD motif is essential for the structural interaction with NKA (Geering et al., 2003; Garty and Karlish, 2006; Geering, 2006). Interestingly, the FXYD motif of AjFXYD11 was present as FXYN harboring an asparagine residue where an aspartate residue is typically found (Fig. 3). This amino acid substitution (D to N) in FXYD11 has also been reported in Atlantic salmon (Tipsmark, 2008) and zebrafish (Saito et al., 2010). Therefore, to realize the functional effect of FXYD11 is necessary in the future works. Then it will be interesting to examine the effect of this amino acid substitution in the FXYD motif on modulation of NKA properties. Moreover, to determine whether AjFXYD11 expression occurs exclusively in the gills, mRNA expression of AjFXYD11 in various tissues was investigated. RT-PCR results showed that AjFXYD11 was expressed exclusively in eel gills (Fig. 4A), which is similar to previous findings in zebrafish (Saito et al., 2010). The real-time PCR, a more sensitive transcript detection technique, was used in previous studies of Atlantic salmon (Tipsmark, 2008) and tilapia (Tipsmark et al., 2010) to examine the expression levels of FXYD11 in various tissues. Using the real-time PCR, we detected AjFXYD11 mRNA in all examined tissues and the branchial abundance of AjFXYD11 transcript was the highest (approximately 130-fold) among all examined organs (Fig. 4B). The results suggest that teleost FXYD11 likely has a conserved function in gills. Furthermore, the abundance of branchial AjFXYD11 was salinity-dependent and increased in parallel with NKA activity in SW-acclimated eel (Fig. 5A). Similar findings have been reported in Atlantic salmon, where the protein amounts of FXYD11 and NKA α-subunit were simultaneously elevated in gills of SW-acclimated individuals (Tipsmark et al., 2010). Saito et al. (2010) used antisense morpholino oligonucleotides targeting FXYD11 to show that FXYD11 expression correlated with NKA expression in the zebrafish. Additionally, the colocalization and interaction between FXYD11 and NKA has been demonstrated in previous experiments (Saito et al., 2010; Tipsmark et al., 2010). Collectively, these and our results presumed that AjFXYD11 plays an osmoregulatory role involved in interaction with NKA in gills. In the eel kidneys, the mRNA abundance of AjFXYD11 was higher than in all other tissues examined except for the gills (Fig. 4B). However, the levels of renal AjFXYD11 mRNA expression were similar between FW- and SW-acclimated eel (Fig. 5B). In the long-term acclimation, the pattern of AjFXYD11 expression was inconsistent with salinity-dependent NKA activity in eel kidneys. These data indicated that AjFXYD11 may be more involved in interaction with NKA in gills rather than in kidneys of A. japonica. Changes in the osmoregulatory properties of fish gills are elicited by changes in gill specific ion-transport proteins. Previous studies have C.-H. Tang et al. / Comparative Biochemistry and Physiology, Part A 163 (2012) 302–310 found that expression levels of gill-specific ion transport protein expression are largely coordinated by changes in circulating cortisol levels (McCormick, 2001; Sakamoto et al., 2001). Because the evidences have shown that cortisol plays a hypoosmoregulatory role in several teleosts, cortisol is considered to be a classical SW adaptation hormone (McCormick, 2001; Mancera and McCormick, 2007). In the eel, an increase in cortisol levels acts primarily to promote the mechanisms of SW adaptation (McCormick, 2001). Moreover, the absence of cortisol via interrenalectomy or hypophysectomy results in a reduction in the activities of gill NKA (McCormick, 2001). In our study, branchial AjFXYD11 expression was elevated in eel acclimated to SW. Furthermore, since in vitro organ culture showed that AjFXYD11 mRNA levels in gills treated in vitro with cortisol were higher than the control, cortisol would play an important role in maintaining gill AjFXD11 mRNA levels (Fig. 6). Gill FXYD11 expression was higher in cortisol treatment has also been found in the Atlantic salmon (Tipsmark et al., 2010) and tilapia (Tipsmark et al., 2011). The cortisol-dependent regulation of FXYD11 expression probably indicates the fact that FXYD11 is most closely related to the mammalian corticosteroid hormone-induced factor, FXYD4 (Tipsmark, 2008). Thus, we propose that cortisol might participate in modulation of the correlation between AjFXYD11 and NKA through maintaining gill AjFXYD11 levels when A. japonica are exposed to SW. NKA is the major energizer of ion-transporting systems in osmoregulatory tissues of teleosts. Therefore, FXYD proteins may be expected to play an important role in osmoregulation of these tissues. By analyzing the effects of environmental salinity and cortisol on FXYD11 expression, the present study provides evidence showing a tight correlation between gill NKA and AjFXYD11 in the catadromous species, A. japonica. Alteration of branchial and renal NKA expression and activity, and gill AjFXYD11 expression in response to FW and SW acclimation showed their close relationship to physiological homeostasis such as stable plasma osmolality and muscle water contents (Fig. 7). Moreover, this study is the first to examine and compare NKA expression and activity in relation to FXYD11 expression in the gills and kidneys simultaneously to illustrate the tissue specificity of FXYD in fish. Future investigations should focus on the role of different FXYD proteins in the regulation of NKA in osmoregulatory tissues to determine whether kidney-specific FXYD genes are expressed in teleosts and the time-course experiments will be conducted to explore the transient changes of AjFXYD11 expression in the Japanese eel. Acknowledgments The monoclonal antibody α5 was purchased from the Developmental Studies Hybridoma Bank (DSHB) maintained by the Department of Pharmacology and Molecular Sciences, John Hopkins University School of Medicine, Baltimore, MD, and the Department of Biological Sciences, University of Iowa, Iowa City, IA, under Contract N01-HD-6-2915, NICHD, USA. This study was supported in part by a grant from the National Science Council (NSC 99-2321-B-005-013-MY3) and in part from the ATU plan of the Ministry of Education, Taiwan (No. 100 S0901), to T.-H.L. Appendix A. 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Na+/K+–ATPase expression in gills of the euryhaline sailfin molly, Poecilia latipinna, is altered in response to salinity challenge. J. Exp. Mar. Biol. Ecol. 375, 41–50. H s FXYD 7 34 Ss FXYD 7 b 98 Ss FXYD 7 a H s FXYD 2 hs FXYD8 81 H s FXYD 6 22 13 Ss FXYD8 34 Ss FXYD 6 95 20 100 Ss FXYD 12 c Ss FXYD 12 b Ss FXYD 1 2a 10 100 20 Ss FXYD 1 1b s s FXYD 11a 75 D rFXYD 11 93 A jFXYD 11 H s FXYD 5 32 Ss FXYD 9b 100 30 Ss FXYD 9a 14 100 Ss FXYD 5b Ss FXYD 5a 76 Ss FXYD2 H s FXYD 4 87 H s FXYD 3 24 H s FXYD 1 PmFXYD 0.1 Fig. S1. Phylogenetic analysis of FXYD proteins based on amino acid sequences using the neighbor-joining method. The tree was generated using FXYD protein sequences from human [Homo sapiens (Hs)], zebrafish [Danio rerio (Dr)], Atlantic salmon [Salmo salar (Ss)] and Japanese eel [Anguilla japonica (Aj)]. A sea lamprey [Petromyzon Marinus (Pm)] member of the gene family was used as the outgroup (accession no. CO551377). Numbers represent bootstrap values in percentage of 1,000 replicates. The accession numbers of the sequences used are as follow: HsFXYD1, AAH32800; HsFXYD2, EAW67326; HsFXYD3, NP_001129483; HsFXYD4, NP_775183; HsFXYD5, NP_001158077; HsFXYD6, NP_001158303; HsFXYD7, NP_071289; HsFXYD8, P58550; DrFXYD11, XP_003200301; SsFXYD2, BK006252; SsFXYD5a, BK006253; SsFXYD5b, BK006254; SsFXYD6, BK006241; SsFXYD7a, BK006245; SsFXYD7b, BK006246; SsFXYD8, BK006242; SsFXYD9a, BK006243; SsFXYD9b, BK006244; SsFXYD11a, NP_001117201; SsFXYD11b, NP_001117200; SsFXYD12a, BK006249; SsFXYD12b, BK006250; SsFXYD12c, BK006251 and AjFXYD11, AFK24487.