Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer

Graham R. Scott,1 Jeff G. Richards,1 Biff Forbush,2,3 Paul Isenring,2,3,4 and Patricia M. Schulte1

1Department of Zoology, University of British Columbia, Vancouver, Canada V6T 1Z4; 2Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510; 3Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672; and 4Groupe de Recherche en Néphrologie, Department of Medicine, Faculty of Medicine, Laval University, Quebec, Canada G1R 2J6

Submitted 27 January 2004 ; accepted in final form 20 March 2004


    ABSTRACT
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 ABSTRACT
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Maintenance of ion balance requires that ionoregulatory epithelia modulate ion flux in response to internal or environmental osmotic challenges. We have explored the basis of this functional plasticity in the gills of the euryhaline killifish Fundulus heteroclitus. The expression patterns of several genes encoding ion transport proteins were quantified after transfer from near-isosmotic brackish water [10 parts/thousand (ppt)] to either freshwater (FW) or seawater (SW). Many changes in response to SW transfer were transient. Increased mRNA expression occurred 1 day after transfer for Na+-K+-ATPase-{alpha}1a (3-fold), Na+-K+-2Cl-cotransporter 1 (NKCC1) (3-fold), and glucocorticoid receptor (1.3-fold) and was paralleled by elevated Na+-K+-ATPase activity (2-fold). The transient increase in NKCC1 mRNA expression was followed by a later 2-fold rise in NKCC protein abundance. In contrast to the other genes studied in the present work, mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel generally remained elevated (2-fold) in SW. No change in protein abundance was detected, however, suggesting posttranscriptional regulation. The responses to FW transfer were quite different from those to SW transfer. In particular, FW transfer increased Na+-K+-ATPase-{alpha}1a mRNA expression and Na+-K+-ATPase activity to a greater extent than did SW transfer but had no effect on V-type H+-ATPase expression, supporting the current suggestion that killifish gills transport Na+ via Na+/H+ exchange. These findings demonstrate unique patterns of ion transporter expression in killifish gills after salinity transfer and illustrate important mechanisms of functional plasticity in ion-transporting epithelia.

Na+-K+-ATPase; Na+-K+-2Cl cotransporter; cystic fibrosis transmembrane conductance regulator; glucocorticoid receptor; H+-ATPase


ION TRANSPORT IS AN ESSENTIAL FUNCTION performed by epithelia in many animal tissues. Inherent to the function of ion-transporting epithelia is the ability to modulate ion flux in response to changing internal and environmental conditions. For example, the euryhaline killifish Fundulus heteroclitus experiences routine fluctuations in environmental salinity in its natural habitat, during which the gill epithelium rapidly adjusts ion flux rates to help maintain ion balance (62). This allows killifish to tolerate salinities ranging from freshwater (FW) to nearly four times seawater (SW) (11). Gill ion transport isfacilitated by mitochondria-rich (MR) cells in the gill epithelium that can individually transform between ion absorption and ion secretion states in response to salinity change (21). Such plasticity makes the gills of killifish an excellent model for investigating how epithelia alternate between absorptive and secretory functions.

The principal transporters responsible for ion movement across gill epithelia have been the subject of numerous reviews (9, 30, 42, 49, 63). Similar to the mechanisms described for several other secretory epithelia (2, 39, 48), ion secretion by fish gills requires a basolateral Na+-K+-ATPase, which creates an electrochemical gradient favoring ion movement, a basolateral Na+-K+-2Cl cotransporter (NKCC), and an apical Cl channel (cystic fibrosis transmembrane conductance regulator, CFTR). Ion absorption by fish gills is not as well understood but likely involves a basolateral Na+-K+-ATPase, either an apical V-type H+-ATPase coupled to a Na+ channel or an apical Na+/H+-exchanger (NHE), and, in some species, an apical Cl/HCO3 anion exchanger (AE).

The ability of killifish to move between SW and FW environments requires modulation of ion flux across the gills, and this is partly controlled by changes in the activity of ion transporters. Short-term regulation of ion transporter activity (and thus ion flux) appears to involve changes in cell volume. For example, an increase in Cl secretion in SW is mediated by cell shrinkage (PKC regulation of NKCC) and cAMP (PKA regulation of CFTR) (14) and involves trafficking of ion transporters to their respective membranes (35). On the other hand, a rapid reduction of Cl secretion in FW is mediated by cell swelling (tyrosine kinase inhibition of CFTR) (32) and sympathetic innervation via {alpha}2-adrenergic receptors (acting through Ca2+/inositol trisphosphate pathway) (31, 33).

In contrast to short-term regulation, relatively few studies have assessed transcriptional and translational regulation of ion transporters after salinity transfer in killifish (4, 56). Such regulatory mechanisms nevertheless appear important. For example, CFTR mRNA expression is known to be upregulated in the gills of this species after transfer from FW to SW (56). Because the killifish is a common model for studying short-term regulation of ion transport by gill epithelia, a good understanding of the integrated responses to salinity transfer can be obtained by studying these additional regulatory aspects. Furthermore, knowledge of the mechanisms underlying modulation of ion fluxes during changing osmotic requirements may lead to the identification of conserved patterns applicable to other ion-transporting epithelia.

The objective of this study was to examine mRNA expression patterns and protein activity or abundance of several ion transporters in killifish gills after salinity transfer. To accomplish this, we transferred fish from near-isosmotic brackish water [BW, 10 parts/thousand (ppt)] to either FW or SW. Isosmotic BW, at which the gradients favoring passive ion flux are minimized, is the preferred salinity for F. heteroclitus (10), and transfer from BW to either extreme of salinity may be more environmentally representative of the conditions killifish naturally encounter in estuaries.


    MATERIALS AND METHODS
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Experimental Animals

Adult killifish (F. heteroclitus macrolepidotus) were captured from estuaries in Hampton, NH (for cloning and salinity transfer) or in the region of Mount Desert Island, ME (for cloning). For the salinity transfer studies, fish were held in static filtered glass aquaria filled with synthetic BW (10 ppt; Deep Ocean, Energy Savers) made up in dechlorinated Vancouver city tap water ([Na+] = 0.17 mM; hardness = 30 mg/l as CaCO3; pH = 5.8–6.4). Before sampling, fish were maintained for at least 30 days in these aquaria at an ambient temperature of 24°C and a 14:10-h light-dark photoperiod. Fish were fed commercial trout chow (PMI Nutrition International) daily. All animal care and experimentation were conducted according to University of British Columbia animal care protocol no. A01-0180.

Total RNA Extraction, Reverse Transcription, and Cloning

Genes of interest were cloned from multiple killifish tissues, including gill, liver, heart, kidney, and spleen, which were sampled after rapid decapitation of the fish. For most cloning experiments, total RNA was extracted from tissues (~20 mg) by using Tripure isolation reagent (Boehringer Mannheim), following the manufacturer's instructions. RNA concentrations were determined spectrophotometrically, and RNA integrity was verified by agarose gel electrophoresis [~1% (wt/vol) agarose-TAE (40 mM Tris-acetate, 2 mM EDTA)]. Extracted RNA samples were stored at –80°C after isolation. First-strand cDNA was synthesized by reverse transcribing 3 µg of total RNA using 10 pmol of oligo(dT18) primer and 20 units of RevertAid H Minus M-MuLV reverse transcriptase (MBI Fermentas), following the manufacturer's instructions.

For some of the cloning experiments, poly(A) RNA was isolated directly using an alternate method (17). Briefly, tissue was homogenized and digested for 1 h in 200 mg/l proteinase K (0.5% SDS, 100 mM NaCl, 20 mM Tris-Cl, and 1 mM EDTA, pH 8.0) at 37°C. After [NaCl] was adjusted to 400 mM, tissue homogenates were incubated with oligo(dT) cellulose for 4 h at room temperature. Poly(A) RNA was eluted in 1 mM EDTA and 0.05% SDS, pH 8.0, and concentrated by ethanol precipitation. In these experiments, the template was primed with a gene-specific antisense oligonucleotide and was extended with the avian myeloblastosis virus (AMV) enzyme and 0.4 µM dNTPs in an appropriate reaction buffer.

Several genes of potential importance for ion regulation in fish were cloned using a PCR-based approach. Multiple alignments of previously published cDNA sequences were constructed using ClustalW (57) to identify conserved gene regions from which primers were then designed (Table 1). Specific sequences within Na+-K+-ATPase-{alpha}1b, glucocorticoid receptor (GR), band 3 anion exchanger 1 (AE1), elongation factor 1{alpha} (EF1{alpha}), NKCC1, and NKCC2 were amplified from killifish gill (Na+-K+-ATPase-{alpha}1b, GR, and NKCC1), liver (EF1{alpha}), spleen (AE1), heart (NKCC1), or kidney (NKCC1 and NKCC2) by using Taq polymerase (MBI Fermentas) or a combination of Taq and Pwo (Invitrogen). Each PCR consisted of 30–40 cycles of 30–40 s at 94°C, 30–50 s at the lower annealing temperature for each respective primer set (see Table 1), and 60 s for every 1,000 bp of expected product at 72°C. PCR products were verified by electrophoresis on 1% agarose gels containing ethidium bromide and were cloned into a pGEM-T Easy (Promega) or pCR2.1 (Invitrogen) vector plasmid. Multiple clones of each fragment were sequenced bidirectionally, and consensus sequences were submitted to the GenBank database (Na+-K+-ATPase-{alpha}1b, accession no. AY430089; GR, accession no. AY430088; AE1, accession no. AY430090; EF1{alpha}, accession no. AY430091; NKCC1, accession no. AY533706; NKCC2, accession no. AY533707). The other killifish gene sequences used in this work were from Na+-K+-ATPase-{alpha}1a (accession no. AY057072), Na+-K+-ATPase-{alpha}2 (accession no. AY057073), CFTR Cl channel (accession no. AF000271), and V-type H+-ATPase subunit A (V-ATPase; accession no. AB066243).


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Table 1. Primers used for cloning of ion transport genes

 
Salinity Transfer Protocol

After at least 1 mo of acclimation to a salinity of 10 ppt, eight control fish were sampled, and those remaining were quickly transferred by net to aquaria containing FW (0 ppt), BW (10 ppt), or SW (35 ppt). Individual fish were subsequently sampled by netting at 3 h, 8 h, 24 h, 96 h, 14 days, and 30 days after transfer from BW. Blood samples were collected in heparinized capillary tubes from the severed caudal peduncle, and fish were killed by rapid decapitation. Blood was centrifuged at 13,000 g for 10 min, and plasma was frozen in liquid nitrogen. The second and third gill arches were isolated and immediately frozen in liquid nitrogen. Gills were not perfused before being frozen because blood has previously been shown to contribute little to whole gill gene expression (43). All tissues were stored at –80°C until analyzed.

Real-Time PCR Analysis of Gene Expression

mRNA was extracted and reverse transcribed from killifish gills by using the first method of reverse transcription described in Total RNA extraction, reverse transcription, and cloning. Gene expression was assessed using quantitative real-time PCR (qRT-PCR) on an ABI Prism 7000 sequence analysis system (Applied Biosystems). Primers for all genes were designed using Primer Express software (version 2.0.0, Applied Biosystems; see Table 2). PCR reactions contained 1 µl of cDNA, 4 pmol of each primer, and Universal SYBR green master mix (Applied Biosystems) in a total volume of 21 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 (set annealing temperature of all primers). PCR products were subjected to melt-curve analysis, and representative samples were electrophoresed to verify that only a single product was present. Control reactions were conducted with no cDNA template or with non-reverse-transcribed RNA to determine the level of background or genomic DNA contamination, respectively. Genomic contamination was less than 1 in 49 starting cDNA copies for all templates except AE1, NKCC2, and Na+-K+-ATPase-{alpha}2 genes. The latter were not detected above background in gills (data not shown) and were subjected to no further analysis. Negligible expression of AE1 in gill samples likely indicates low relative contamination by erythrocytes, in which AE1 is highly expressed in fish (15).


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Table 2. Primers used for qRT-PCR of ion transport genes

 
A randomly selected control sample was used to develop a standard curve for each primer set, and all results were expressed relative to these standard curves. Results were then normalized to EF1{alpha}, a gene for which mRNA expression in the gills does not change after salinity transfer (data not shown). For clarity in graphing, posttransfer samples were expressed relative to the pretransfer BW control samples. All samples were run in duplicate (coefficients of variation were ≤10%). To avoid unnecessary analysis, we analyzed pretransfer controls and 24 h, 96 h, and 14 day time points first. Expression at remaining time points was analyzed only for those genes that were affected consistently by changes in salinity.

The absolute level of mRNA expression of each gene examined was estimated semiquantitatively 24 h after transfer (BW, FW, and SW) according to the following formula:

(1)
where efficiency is determined from the slope of the standard curve and Ct corresponds to the threshold cycle number. These results were subsequently normalized to the estimated absolute expression of EF1{alpha}. Because of the nature of analysis using qRT-PCR, these data can only be used for approximate comparison of expression levels between genes.

Na+-K+-ATPase Activity Assay and Western Blot Analysis

Na+-K+-ATPase activity was determined by coupling ouabain-sensitive ATP hydrolysis to pyruvate kinase- and lactate dehydrogenase-mediated NADH oxidation as outlined by McCormick (36). For this assay, the second and third gill arches were homogenized in 500 µl of SEI (150 mM sucrose, 10 mM EDTA, and 50 mM imidazole, pH 7.3) containing 0.1% Na-deoxycholate and centrifuged at 5,000 g for 30 s at 4°C. Supernatants were immediately frozen in liquid nitrogen and stored at –80°C until analyzed. ATPase activity was determined in the presence or absence of 0.5 mM ouabain by using 10 µl of supernatant thawed on ice and was normalized to total protein content (measured using the bicinchoninic acid method; Sigma-Aldrich). All samples were run in triplicate (coefficients of variation were ≤10%). Ouabain-sensitive ATPase activity is expressed as micromoles of ADP per milligram of protein per hour.

NKCC and CFTR protein abundance was measured by Western immunoblotting according to the method of Marshall et al. (35). Gill homogenates were prepared as outlined in Na+-K+-ATPase activity assay and Western blot analysis and were denatured for 3 min in boiling SDS-sample buffer (23). SDS-polyacrylamide gels (8%) were loaded with total gill homogenates (20 µg protein/lane), and protein was transferred to nitrocellulose membranes (Bio-Rad) by using a Trans-Blot semidry transfer cell (Bio-Rad). Blots were first incubated for 1 h with 1.0 µg/ml primary antibody diluted in TTBS buffer (17.4 mM Tris·HCl, 2.6 mM Tris base, 500 mM NaCl, 2.0 mM sodium azide, and 0.05% Tween 20, pH 7.5) containing 2% skim milk; the primary antibody used against killifish NKCC protein was a polyclonal mouse anti-human NKCC (T4; Iowa Hybridoma Bank, University of Iowa) (27), and the primary antibody used against killifish CFTR protein was a monoclonal mouse anti-human CFTR (R&D Systems). Blots were subsequently incubated for 1 h with goat anti-mouse IgG secondary antibody (alkaline phosphatase conjugated; Stressgen) diluted 1:3,000 in TTBS. Membranes were developed in alkaline phosphatase buffer containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) (Sigma-Aldrich). Band intensity was quantified using a FluorChem 8800 imager (Alpha Innotech) assisted by AlphaEaseFC software (version 3.1.2; Alpha Innotech). Samples were expressed relative to a randomly chosen protein standard (included on each gel to control for transfer efficiency) and normalized to pretransfer BW control samples. In these studies, the sensitivity of Western immunoblotting for quantification of protein content was verified through dose-response analyses of both antibodies by using homogenates from two fish.

Plasma Variables

Plasma Na+ was determined using flame atomic absorption spectrophotometry (SpectrAA-220FS; Varian) with Fisher certified standards. Plasma cortisol was determined using enzyme-linked immunosorbent assay (Neogen), following the manufacturer's instructions.

Statistical Analyses

Data are expressed as means ± SE. For gene expression, protein abundance, transporter activity, and plasma variables, Friedman's two-factor, nonparametric analysis of variance (ANOVA) was used to determine whether the effect of salinity over time differed among BW, SW, and FW. Kruskal-Wallis H nonparametric ANOVA was used to ascertain overall differences as a function of either salinity (within each time point) or time (for each salinity). Because measured variables could have changed as a result of handling of fish alone, the effect of SW or FW transfer was assessed by comparison with the matched BW control, using Mann-Whitney U nonparametric comparisons at time points at which salinity effects were detected by ANOVA. All statistical analyses were conducted using SPSS version 10.0, and a significance level of P < 0.05 was used.


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Plasma Na+

Changes in plasma Na+ as a function of salinity over time were significantly different among salinity groups (Fig. 1). Plasma Na+ did not change relative to matched BW controls at any time point sampled after transfer to SW. In contrast, transfer to FW decreased plasma Na+ compared with BW controls 1 day after transfer.



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Fig. 1. Plasma Na+ (n ≥ 5) in killifish before (pre) and after transfer from brackish water [BW, 10 parts/thousand (ppt)] to BW, seawater (SW), or freshwater (FW). Data are expressed as means ± SE. *Significant difference from time-matched BW control (P < 0.05).

 
Expression of Genes in Killifish Gills

Estimated absolute mRNA expression level (determined using Eq. 1, measured 1 day after transfer to all salinities) followed the order from highest to lowest (shown relative to EF1{alpha} in parentheses): Na+-K+-ATPase-{alpha}1a (10–1), NKCC1 (10–2), Na+-K+-ATPase-{alpha}1b (10–2), GR (4 x 10–3), V-ATPase (2 x 10–3), and CFTR (3 x 10–4). For most of these genes, changes in mRNA expression, protein abundance, or transporter activity as a function of salinity over time were significantly different among the three salinity groups (see Figs. 25). In addition, most of these variables also changed significantly as a function of time within each salinity group, including the BW controls. For this reason, the data reported focus on the effect of SW or FW transfer compared with BW controls within each time point.



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Fig. 2. Na+-K+-ATPase-{alpha}1a mRNA expression (n ≥ 7) (A) and Na+-K+-ATPase activity (n ≥ 6) (B) in killifish gills before (pre) and after transfer from BW to BW, SW, or FW. Expression is relative to expression of elongation factor 1{alpha} (EF1{alpha}) and is normalized to pretransfer controls. FW activities are offset for clarity at 3, 8, and 24 h. Data are expressed as means ± SE. *Significant difference from time-matched BW control (P < 0.05).

 


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Fig. 5. Plasma cortisol (n ≥ 6) (A) and glucocorticoid receptor (GR) mRNA expression in gills (n ≥ 7) (C) in killifish before (pre) and after transfer from BW to BW, SW, or FW. FW data are offset for clarity at 3 and 8 h. Expression is relative to expression of EF1{alpha} and is normalized to pretransfer controls. Data are expressed as means ± SE. *Significant difference from time-matched BW control (P < 0.05).

 
Na+-K+-ATPase. Patterns of Na+-K+-ATPase-{alpha}1a mRNA expression in the gills differed between SW and FW transfers (Fig. 2A). When fish were transferred from BW to SW, Na+-K+-ATPase-{alpha}1a expression increased transiently 1 day after transfer, to nearly threefold higher than in BW controls. There was also a small but statistically significant increase at 14 days. In contrast to SW, the increase in Na+-K+-ATPase-{alpha}1a mRNA expression in fish transferred from BW to FW was more prolonged, peaking sixfold higher than in BW controls at 4 days (Fig. 2A). In fact, the increase in expression 4 days after FW transfer was higher than at any point in SW. Interestingly, although mRNA of the Na+-K+-ATPase-{alpha}1b isoform was expressed at high levels (see above), expression did not change in SW and was only slightly decreased (0.8-fold) 14 days into FW (Table 3).


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Table 3. Gill mRNA expression measured by qRT-PCR after salinity transfer

 
Na+-K+-ATPase activity changed in a manner similar to Na+-K+-ATPase-{alpha}1a expression after both SW and FW transfer (Fig. 2B). A transient twofold increase in Na+-K+-ATPase activity was observed 1 day into SW compared with matched BW controls. Na+-K+-ATPase activity underwent a more prolonged increase after FW transfer, rising twofold higher than in BW controls from 1 to 4 days.

NKCC. NKCC1 mRNA expression patterns in the gills were very similar to those for Na+-K+-ATPase-{alpha}1a after SW transfer, showing comparable transient changes (Fig. 3A). Expression of this gene increased threefold higher than in BW controls 1 day after transfer, with a small subsequent rise at 14 days. In contrast, NKCC1 mRNA expression decreased 2.5-fold 1 day into FW.



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Fig. 3. Na+-K+-2Cl-cotransporter 1 (NKCC1) mRNA expression (n ≥ 7) (A) and NKCC protein abundance (n ≥ 6) (C) in killifish gills before (pre) and at several times after transfer from BW to BW, SW, or FW. Expression is relative to expression of EF1{alpha} and is normalized to pretransfer controls. B: representative Western blots at 14 days. Protein abundance was quantified at ~170 kDa by using mouse anti-human NKCC antibody (see MATERIALS AND METHODS for details). This molecular mass corresponds to fully glycosylated NKCC protein. Data are expressed as means ± SE. *Significant difference from time-matched BW control (P < 0.05).

 
Western blot analyses performed using the T4 antibody (mouse anti-human NKCC) revealed a single 170-kDa immunoreactive band (Fig. 3B), likely corresponding to fully glycosylated NKCC protein (38). NKCC abundance estimated at this band increased twofold 14 and 30 days after SW transfer, but there was no change after FW transfer (Fig. 3C).

CFTR Cl channel. Changes in CFTR mRNA expression were more prolonged than those for Na+-K+-ATPase-{alpha}1a and NKCC1 in SW. CFTR expression increased rapidly 3 h after transfer to SW (2-fold higher than in BW controls) and remained elevated until returned to control levels 30 days later (Fig. 4A). On the other hand, CFTR mRNA expression was generally reduced in FW, decreasing as much as 10-fold less than in BW controls at 1 day posttransfer.



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Fig. 4. Cystic fibrosis transmembrane conductance regulator Cl channel (CFTR) mRNA expression (n ≥ 7) (A) and CFTR protein abundance (n ≥ 6) (C) in killifish gills before (pre) and after transfer from BW to BW, SW, or FW. Expression is relative to expression of EF1{alpha} and is normalized to pretransfer controls. B: representative Western blots at 1 day. Protein abundance was quantified at ~160 kDa by using mouse anti-human CFTR antibody (see MATERIALS AND METHODS for details), corresponding to the molecular mass of mature CFTR protein. A faint immunoreactive band at ~120 kDa was not quantified and likely represents immature unglycosylated protein. Data are expressed as means ± SE. *Significant difference from time-matched BW control (P < 0.05).

 
CFTR abundance on immunoblots was estimated at an approximate size of 160 kDa, corresponding to the mature protein (Fig. 4B). A second, faint band at 120 kDa was not quantified and probably represents immature unglycosylated protein (1). CFTR protein abundance at 160 kDa did not change over time in SW, and no differences from that in matched BW controls were detected (Fig. 4C). FW transfer increased CFTR protein levels at 1 day posttransfer (Fig. 4C).

V-ATPase. There were only minor effects of salinity on V-ATPase mRNA expression (Table 3). SW transfer increased expression above that in BW controls (1.2-fold) 1 day posttransfer, whereas FW transfer had no effect on V-ATPase expression compared with BW controls at any time point.

Plasma cortisol and GR. Plasma cortisol responded in a similar manner to each salinity over time (Fig. 5A). Transfer from BW to BW transiently increased plasma cortisol, probably as a result of handling. Plasma cortisol also increased transiently after both SW and FW transfer, but changes were more pronounced than for matched BW controls. After transfer from BW to SW, plasma cortisol was nearly threefold higher than in BW controls at 3 h. FW transfer resulted in a similar threefold rise in plasma cortisol 3 h after transfer.

GR mRNA expression tended to decrease immediately after transfer to all three salinities, followed by a transient rise in expression at 1 day (Fig. 5B). Despite these changes with time, however, both FW and SW transfer reduced GR expression 8 h after transfer compared with BW controls (1.7-fold less than in BW controls), and SW transfer increased GR expression 1 day posttransfer (1.3-fold higher than in BW controls).

Correlation of Ion Transporter mRNA Expression

After SW transfer, Na+-K+-ATPase-{alpha}1a and NKCC1 mRNA expression were very well correlated (r2 = 0.872, P < 0.001) (Fig. 6A). Expression was related such that a threefold increase in NKCC1 coincided with a twofold increase in Na+-K+-ATPase-{alpha}1a (slope of 1.4 ± 0.1). Na+-K+-ATPase-{alpha}1a and CFTR also were positively correlated in SW, but this relationship was not as strong (r2 = 0.309, P < 0.001; slope of 0.5 ± 0.1) (Fig. 6B). There was a similar association between NKCC1 and CFTR after SW transfer (r2 = 0.202, P < 0.001) (data not shown). Expression of ion transporters after FW transfer was not correlated to the same degree (data not shown): Na+-K+-ATPase-{alpha}1a and CFTR showed a slight negative correlation posttransfer (r2 = 0.186, P = 0.002), but expression of neither gene correlated with NKCC1 (P > 0.448).



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Fig. 6. NKCC1 (A) and CFTR (B) mRNA expression correlated to Na+-K+-ATPase-{alpha}1a mRNA expression in killifish gills at several times after transfer to SW. mRNA expression is relative to expression of EF1{alpha}. A: NKCC1; r2 = 0.872, P < 0.001, y = (1.4 ± 0.1) – (0.7 ± 0.2). B: CFTR; r2 = 0.309, P < 0.001, y = (0.5 ± 0.1) – (1.1 ± 0.2). Hatched lines represent 95% confidence limits of regression.

 

    DISCUSSION
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 MATERIALS AND METHODS
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Killifish typically encounter fluctuations in environmental salinity on a daily basis in their natural habitat. Rapid modulation of ion flux across the gills is thus important for maintaining ion balance and likely occurs through posttranslational processes. These include changes in the subcellular localization and activity of various ion transporters (14, 3133, 35, 59), as well as rapid changes in gill epithelium morphology (7), leading to immediate changes in transepithelial ion currents. The rapid adjustments that occur shortly after salinity change likely allow these animals to maintain ionic homeostasis in the face of fluctuations in estuarine salinity that are transient and somewhat unpredictable. However, salinity change may persist for a longer duration, such as when killifish move up and down tributaries between seasons (12) and when populations invade FW (45). In these situations, additional mechanisms may be important for long-term adjustment to salinity change. For example, prolonged transfer to SW or FW appears to involve changes in the abundance of mitochondria-rich cells in the gill epithelium (21). In the current study, we have also observed unique patterns of ion transporter expression in the gills of killifish after transfer to either SW or FW. Gene regulation and cell proliferation likely contribute to the further increases in ion flux that are observed one or more days after salinity change (62). Our work and that of others therefore demonstrates very clearly that the gills of killifish are able to employ an array of regulatory strategies, including those at transcriptional and translational levels, to regulate ion balance in various environments. As a result, even after large changes in environmental salinity, killifish experience only small, transient changes in plasma ion levels (present study and Refs. 18, 21, 34).

Ion transporter expression after SW transfer. Consistent with the physiological model for ion secretion by gill epithelia, SW transfer increased the expression of Na+-K+-ATPase-{alpha}1a, NKCC1, and CFTR at the mRNA level. The gill epithelium is heterogeneous, so the two- to threefold changes in ion transporter expression we report in the present study in whole gills may have been much higher in individual ion-transporting cells. Interestingly, the increase in expression was transient for Na+-K+-ATPase-{alpha}1a and NKCC1 but prolonged for CFTR. Furthermore, Na+-K+-ATPase-{alpha}1a and NKCC1 expression patterns were tightly correlated after SW transfer (r2 = 0.872), but expression of both genes was poorly correlated (r2 ≤ 0.309) with that of CFTR. Differentially sensitive salt- or hormone-responsive regulatory elements for Na+-K+-ATPase-{alpha}1a, NKCC1, and CFTR genes could explain these results. Alternatively, basolateral (Na+-K+-ATPase-{alpha}1a and NKCC1) and apical (CFTR) ion transporter genes may be regulated by different transcription factors. Future experiments are required to determine the factors regulating transcription of ion transporter genes in killifish gills.

Changes in protein abundance are often assumed to parallel changes in mRNA expression. Consistent with this assumption, Na+-K+-ATPase activity, although not necessarily an indicator of protein abundance, increased in parallel with Na+-K+-ATPase-{alpha}1a mRNA expression. This was not the case for NKCC after SW transfer, however, because increased NKCC1 gene expression preceded a prolonged increase in NKCC protein abundance. The prolonged increase in NKCC protein, coupled with the transient increase in NKCC1 mRNA levels, suggests that NKCC protein turnover in the gill may be low. Parallel changes in CFTR mRNA and protein levels were also absent. In fact, CFTR protein abundance did not change after SW transfer, even though mRNA expression was elevated for a prolonged period. This lack of correlation suggests posttranscriptional regulation of protein abundance. Elevated Cl conductance in SW might therefore be modulated primarily by posttranslational mechanisms (e.g., intracellular trafficking; see Refs. 1, 35) and might not involve changes in CFTR protein abundance. Together, our data suggest that the dynamic between gene transcription and protein abundance is not always straightforward and may depend on numerous factors (see Refs. 8 and 61 for review).

Some similarity exists between the patterns of gene expression reported in the present study for killifish and those reported in previous studies for other species. For example, a prolonged increase in CFTR expression has also been observed in the gills of Atlantic salmon (Salmo salar) transferred from FW to SW (55). Similarly, NKCC protein has been shown to increase in the gills of Atlantic salmon (41) and tilapia (Oreochromis mossambicus) (64) after SW acclimation.

Despite some similarities in gene expression among species, our data also demonstrate that the patterns of gene expression among various fish species are not always the same. For example, Na+-K+-ATPase-{alpha}1a and NKCC1 mRNA expression increase only transiently in the gills of killifish after SW transfer, whereas expression of these genes increases for more prolonged periods in the gills of Atlantic salmon (6, 51, 58), brown trout (Salmo trutta) (28, 58), or European sea bass (Dicentrarchus labrax) (19). Transient changes in killifish gill Na+-K+-ATPase activity after SW transfer were also observed in this study and in others (29, 59), whereas more prolonged increases in Na+-K+-ATPase activity are typical of many fish species (19, 24, 28, 47, 50, 51). The transient nature of many changes observed in gill protein expression and activity appears to be an important response of killifish to SW transfer and may reflect greater tolerance to salinity change. Plasma Na+ did not change significantly after transfer from BW to SW, so prolonged changes in expression may be unnecessary for maintaining ion balance. Different physiological responses of fish to changes in salinity may also have arisen from the effects of alternative life histories (e.g., anadromy vs. euryhalinity) or evolutionary histories (i.e., FW vs. SW ancestry).

Ion transporter expression after FWtransfer. Recent work has suggested that the mechanism of ion uptake by killifish gills in FW differs from the prevalent model, which was developed predominantly in salmonids. In these species it is proposed that Na+ uptake through epithelial Na+ channels (ENaC) is driven by the electrical gradient produced by apical V-ATPase (25, 43, 46). In killifish, however, Patrick and Wood (40) demonstrated that Na+ uptake and H+ efflux in FW is inhibited by both amiloride and low water pH to the same degree, suggesting that apical Na+ flux is primarily mediated by NHEs. Similarly, in a discussion of some unpublished observations, Claiborne et al. (5) noted that NHE2 mRNA expression increased in the gills of killifish after FW transfer. Consistent with these findings, our data show that transcription of V-ATPase was unaffected by salinity transfer, even though it was expressed at high levels in the gills. Together, these data suggest that NHEs may be important for Na+ absorption across the apical membrane of the gills of FW killifish. Interestingly, recent work by Katoh et al. (20) immunolocalized V-ATPase to the basolateral membrane of mitochondria-rich cells in the gills of FW killifish, and immunoreactivity increased in dilute FW. Thus basolateral V-ATPase may play a role in ion absorption, regulated primarily through posttranscriptional mechanisms. FW elasmobranch gills express V-ATPase on the basolateral surface of mitochondria-rich cells as well (44), so in this way the mechanisms of ion transport in the gills of killifish and some elasmobranchs may be similar.

Na+-K+-ATPase-{alpha}1a mRNA expression and Na+-K+-ATPase activity increased after FW transfer, and maximal changes to levels higher than in BW controls coincided with the reestablishment of plasma Na+. Increased Na+-K+-ATPase transcription may occur in conjunction with chloride cell hypertrophy, as previously described for killifish in FW (21, 34). Interestingly, both expression and activity were upregulated to a greater extent after FW transfer than after SW transfer in killifish, similar to findings reported for some other species [e.g., milkfish, Chanos chanos (26)]. In contrast, for many species (e.g., salmonids, tilapia, and others; discussed above), Na+-K+-ATPase mRNA expression and activity increase to their greatest extent after SW transfer. These species also have been reported to upregulate apical V-ATPase in FW (13, 25, 52). The discrepancy in Na+-K+-ATPase regulation between killifish and these species may therefore relate to the above suggestion that apical NHEs mediate Na+ absorption in killifish, rather than ENaC coupled to V-ATPase. Without the electrochemical gradient created by active apical extrusion of protons by V-ATPase, higher Na+-K+-ATPase activity may be required in the gills of killifish to maintain a favorable Na+ gradient across the apical membrane.

As was the case after SW transfer, mRNA expression patterns after FW transfer were not necessarily followed by predictable changes in protein abundance. For example, both NKCC1 and CFTR mRNA expression decreased in killifish gills after FW transfer, whereas protein abundance did not. In fact, CFTR abundance (measured at 160 kDa) actually increased 1 day after FW transfer. A second isoform such as that in Atlantic salmon (55) could account for this increase in CFTR abundance. Our real-time PCR primers may have failed to amplify a second isoform, while our antibody may have cross-reacted with both isoforms. Interestingly, CFTR immunofluorescence in the cytoplasm and/or on the basolateral surface of pavement cells in killifish opercular epithelium increases in FW (detected by the same antibody used in the present study; Ref. 35), possibly indicating that a FW-specific isoform exists.

Cortisol and salinity transfer. Elevation of plasma cortisol is recognized as an important part of the endocrine signaling response that occurs shortly after SW transfer and has been observed in several fish species, including killifish (18, 34). In the present study, we have observed a similar rise in plasma cortisol during the 24 h after SW transfer. This response may in fact account for the upregulation of Na+-K+-ATPase in fish gills that also occurs in SW as suggested by McCormick (37).

In this study, plasma cortisol also increased transiently after FW transfer, as previously reported in killifish (18). In contrast to SW transfer, however, the role of this hormone in fish during the early stages of FW acclimation is poorly understood. Interestingly, cortisol treatment promotes ion uptake both in vivo and across cultured gill epithelia and also has been shown to increase Na+-K+-ATPase activity as well as transepithelial resistance and to reduce passive ion fluxes (22, 37, 65). Therefore, cortisol may have contributed to the Na+-K+-ATPase upregulation observed after FW transfer. To this effect, it is interesting to note that Na+-K+-ATPase mRNA expression is increased by glucocorticoids in some mammalian absorptive tissues, including lung (16) and renal tubule (3) epithelia.

GR expression was observed to decrease early after BW, FW, and SW transfers, during which time plasma cortisol increased. Furthermore, the plasma cortisol elevation observed 3 h after both SW and FW transfers was followed by decreased GR expression at 8 h compared with BW controls. These changes in GR mRNA expression observed over time suggest negative feedback regulation by cortisol, as has been previously suggested for salmonids (54, 60). However, the changes in GR expression may not have been solely due to negative feedback regulation. Increased expression of this receptor occurred after 1 day in SW compared with BW and FW, and this was not preceded by decreased plasma cortisol. Hence, other specific effects of SW transfer could have brought about changes in GR expression. These factors may be better appreciated if the potentially interactive effects of cortisol and osmotic change are eliminated.

The transient rise in GR expression after SW transfer is another interesting result in this study. Indeed, this behavior contrasts with that observed in chum salmon (Oncorhynchus keta), for which GR expression in gills increases progressively with SW acclimation (60). Differences between the responses of anadromous and euryhaline fish to salinity transfer may therefore apply beyond the expression and function of ion transporters to various regulatory systems.

The data presented in the current study indicate that responses to salinity transfer differ between killifish and salmonids. Differences in life histories between these taxa may have contributed to these varied physiological strategies used to cope with variation in environmental salinity. Salmonids are anadromous and generally prepare for migration between salinities at specific stages of their life cycle, so it is perhaps foreseeable that they rely strongly on long-term changes in transcription and translation of ion transporters to maintain ion balance. In contrast, killifish encounter daily variations in estuarine salinity that are somewhat unpredictable, so they regulate ion transporters in various ways, many of which are rapid and/or transient.

In summary, we have quantified the expression patterns of several ion transport genes in the gills of killifish after transfer from intermediate salinity to FW and SW; such transfers are probably representative of what many estuarine fish normally encounter in their natural habitats. Interestingly, we observed that the expression patterns varied appreciably as a function of salinity and that changes in mRNA expression were not always matched by changes in protein abundance. This work advances our understanding of the factors regulating ion transport in euryhaline fish and demonstrates important mechanisms of functional plasticity in transport epithelia.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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This work was funded by the Natural Sciences and Engineering Research Council of Canada through a Discovery Grant (to P. M. Schulte), a Post Graduate Scholarship (to G. R. Scott), and a Post Doctoral Fellowship (to J. G. Richards), as well as an Izaak Walton Killam Post Doctoral Fellowship (to J. G. Richards). This work was also supported by National Institutes of Health Grants DK-47661 and ES-3828 (to B. Forbush), Canadian Institutes of Health Research and Kidney Foundation of Canada grants (to P. Isenring), and by the Mount Desert Island Laboratory (Salsbury Cove, Maine).


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. R. Scott, Dept. of Zoology, Univ. of British Columbia, Vancouver, BC, Canada V6T 1Z4 (E-mail: scott{at}zoology.ubc.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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