Na+/K+-ATPase -isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer
1 Department of Zoology, The University of British Columbia, Vancouver,
British Columbia, Canada, V6T 1Z4
2 Department of Biology, University of Waterloo, Waterloo, Ontario, Canada
N2L 3G1
3 Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G
2W1
Author for correspondence (e-mail:
jrichard{at}zoology.ubc.ca)
Accepted 2 September 2003
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Summary |
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Key words: gene expression, seawater, freshwater, ion regulation, salmonid, rainbow trout, Oncorhynchus mykiss, Na+/K+-ATPase
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Introduction |
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Na+/K+-ATPase is a membrane protein that couples the
exchange of two extracellular K+ ions for three intracellular
Na+ ions to the hydrolysis of one molecule of ATP
(Mobasheri et al., 2000). The
functional Na+/K+-ATPase is composed of two essential
subunits (
and ß) that are noncovalently paired to form an
ß-heterodimer. The
-subunit contains the binding sites for
ATP, Na+, K+ and the cardiac glycoside, ouabain, and is
considered the catalytic subunit. The ß-subunit is a type II glycosylated
polypeptide that is thought to assist in the folding and placement of the
-subunit into the cell membrane
(Blanco and Mercer, 1998
). A
third, nonessential
-subunit has been identified in mammals
(Reeves et al., 1980
), and is
thought to play a role in modulating Na+-, K+- and
ATP-binding affinities to the Na+/K+-ATPase
ß complex (Béguin et
al., 1997
; Therien et al.,
1999
). Thus far, four
-, three ß- and one
-subunit isoforms have been identified in mammals
(Mobasheri et al., 2000
) and
several
- and ß-subunits have been identified in fish. Full or
partial cDNA sequences for Na+/K+-ATPase
1-,
2- and
3-isoforms have been identified in numerous fish species
including salmonids (Cutler et al.,
1995a
,b
;
Guynn et al., 2002
;
Schonrock et al., 1991
;
Semple et al., 2002
); however,
little is known about the physiological role of these isoforms.
Among the four Na+/K+-ATPase -isoforms
identified in mammals, differences in Na+, K+ and ATP
binding affinity and sensitivity to ouabain have been described.
Characterization of rat Na+/K+-ATPase
-and
ß-isoforms in insect cell lines demonstrated that Na+ and
K+ affinity varied among Na+/K+-ATPase
isoform combinations with a rank order of
2ß1>
1ß1>
3ß1 and
1ß1>
2ß1>
3ß1, respectively (see
Blanco and Mercer, 1998
). Thus,
differences in the
-isoforms strongly influence the kinetic properties
of Na+/K+-ATPase and the tissue distribution of these
isoforms may be related to matching isoform-specific kinetic properties with
tissue-specific physiological functions. Indeed, the physiological role of
these isoforms have recently been examined in several mammalian tissues (e.g.
He et al., 2001
;
James et al., 1999
;
Woo et al., 2000
). However,
little is known about the importance of differential
Na+/K+-ATPase
-isoform expression in organs such
as fish gills that must dynamically regulate ion balance.
There is accumulating evidence to suggest that fish gills express multiple
Na+/K+-ATPase -isoforms and that the isoform
complement may change to meet the ion-regulatory challenges imposed during
salinity transfer. Pagliarani et al.
(1991
) demonstrated that
Na+/K+-ATPase proteins isolated from freshwater- and
seawater-acclimated fish gills had different biochemical properties,
suggesting that different Na+/K+-ATPase isoforms may be
involved in freshwater versus seawater ion regulation. Furthermore,
Lee et al. (1998
) showed
increases in Na+/K+-ATPase
1- and
3-protein abundance in the gills of tilapia Oreochromis
mossambicus following transfer from freshwater to seawater. Analysis of
Arrhenius plots also provides evidence for the presence of multiple
Na+/K+-ATPase isoforms in carp Cyprinus carpio
gills and implicates these isoforms in thermal acclimation
(Metz et al., 2003
). Overall,
changes in Na+/K+-ATPase
-isoform expression may
be a crucial mechanism to match the kinetic properties of
Na+/K+-ATPase to changing environmental conditions.
Thus, the objectives of the present study were to determine if multiple
Na+/K+-ATPase -isoforms exist in rainbow trout
and to examine the expression pattern of these isoforms in trout gills
following abrupt transfer from freshwater to 40% and 80% seawater.
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Materials and methods |
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Identification and sequencing of Na+/K+-ATPase
isoforms
Tissue sampling
Trout were sampled directly from the holding tank and killed by
decapitation. Samples of brain, gill, eye, heart, liver, spleen, intestine,
kidney, testis, white muscle and red muscle were dissected from the trout and
immediately frozen in liquid N2. Tissue samples were stored at
70°C until analysis.
RNA extraction and reverse-transcriptase PCR amplification
Total RNA was extracted from tissues using the guanidine thiocyanate method
outlined by Chomczynski and Sacchi
(1987) using TriPure Isolation
Reagent (Roche Diagnostics, Montreal, QC, Canada). Following isolation, RNA
was quantified spectrophotometrically and electrophoresed on an
agaroseformaldehyde gel (1.2% w/v agarose, 16% formaldehyde) to verify
RNA integrity. RNA was stored at 80°C.
First strand cDNA was synthesized from 4 or 5 µg of total RNA isolated
from the above tissues using oligo(dT15) primer and
RevertAidTM H Minus M-MuLV reverse transcriptase, following the
manufacturer's instructions (MBI Fermentas Inc., Burlington, ON, Canada).
Partial Na+/K+-ATPase -subunit sequences were
obtained using primers determined from conserved regions of Torpedo
californica (Accession No. X02810), Catostomus commersoni
(Accession No. X58629), Anguilla anguilla (Accession No. X76108), and
Xenopus laevis (Accession No. U10108). The forward primer (NaKF) was
5'-AAC CCC AGA GAT GCC AA-3' and the reverse primer (NaKR) was
5'-AAG GCA CAG AAC CAC CA-3' (see also
Semple et al., 2002
). Primers
were designed with the assistance of GeneTool Lite software
(www.biotool.com).
Polymerase chain reactions (PCRs) were carried out in a PTC-200 MJ Research
themocycler using Taq DNA polymerase (MBI Fermentas) and cDNA
isolated from the above tissues. Each PCR consisted of 35 cycles: 1 min at
94°C, 1 min at 42°C and 2 min at 72°C. PCR products were
electrophoresed on 1% agarose gels containing ethidium bromide and bands of
appropriate size were extracted from the gels using QIAEX II gel extraction
kit (Qiagen Inc., Mississauga, ON, Canada). The extracted PCR product from
each tissue was cloned into a T-vector (pGEM-T easy; Promega; Fisher
Scientific, Nepean, ON, Canada), transformed into heat shock competent
Escherichia coli (strain JM109; Promega, Fisher Scientific, Nepean,
ON, Canada) and colonies grown on ampicillin LB-agar plates. Several colonies
containing the ligated PCR product were selected and grown overnight in liquid
culture. Plasmids were harvested from liquid culture using GenElute Plasmid
Miniprep kit (Sigma-Aldrich, Oakville, ON, Canada) and sequenced using an ABI
377 automated fluorescent sequencer at York University Molecular Biology core
facility (Toronto, ON, Canada).
Isoform-specific primers were designed to obtain the complete cDNA sequence
using 5' and 3' RACE (Smart RACE cDNA amplification kit; BD
Bioscience Clontech, Mississauga, ON, Canada). Multiple clones of each
fragment were sequenced in both directions at least twice, and a majority-rule
consensus for the full-length cDNA transcript was developed for each isoform.
Sequence assembly and analysis were performed using GeneTool Lite software.
Comparison with published sequences in GenBank was made with the BLAST
algorithm (Altschul et al.,
1997) and multiple alignments were produced with ClustalW
(Thompson et al., 1994
).
Putative transmembrane regions were identified by hydrophobicity analysis
using AnTheProt
(http:\antheprot-pbil.ibcp.fr/ie_sommaire.html).
cDNA sequences have been deposited into GenBank with accession numbers given
in Table 1.
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Tissue distribution
The distribution pattern of Na+/K+-ATPase
-subunit mRNA was determined using isoform-specific PCR and ethidium
bromide stained gels. Alignment of the Na+/K+-ATPase
-isoform sequences were used to determine regions that were unique to
each isoform and PCR primers were designed in these areas. PCR reactions were
performed with the isoform-specific primers and cDNA obtained from the
dissected tissues.
Phylogenetic analysis
Amino acid (aa) sequences were deduced from the nucleotide sequence of each
isoform using GeneTool Lite software. Protein sequences or deduced aa sequence
of other Na+/K+-ATPase -subunits were obtained
from GenBank: human
1 (X04297); human
2 (XP010502); human
3 (NM000703); human
4 (Q13733); rat
1 (D00189), rat
2 (NM012505), rat
3 (NP036638); rat
4 (NP074039); chicken
1 (J03230); chicken
2 (17041294); chicken
3 (P24798);
mouse
4 (Q9WV27); horse
1 (P18907); sheep
1 (P04074); dog
1 (P50997); pig
1 (P05024); killifish
1 (AY057072);
killifish
2 (AY057073); tilapia
1 (U82549); tilapia
3
(AF109409); electric eel (AF356351); frog (P30714); goldfish
3
(BAB60722); Torpedo ray (P05025); whitesucker (X58629), eel (Q92030); Xenopus
1 (Q92123); zebrafish
1 (AF286372); zebrafish
2
(AF286373); zebrafish
3 (AF286374); zebrafish
4 (AF308598);
zebrafish
5 (AF308599); zebrafish
6 (AY008374); zebrafish
7 (AY008375); zebrafish
8 (AY008376); fruitfly (AAF55826).
Sequences were aligned using ClustalW and phylogenetic analysis was performed
using the neighbour-joining method with complete deletion of gaps using MEGA2
software (Kumar et al., 2001
).
The support for each node was assessed using 500 bootstrap replicates.
Isoforms were named according to their position in the phylogenetic tree.
Sliding window analysis
To determine the percentage variation between the cDNA and aa sequences of
the five Na+/K+-ATPase -isoforms, we performed
sliding window analysis. Sliding window analysis quantifies the variation
between aligned sequences by counting the average number of differences
between isoforms within overlapping windows. For the present analysis, we used
an overlapping window of 50. Sliding window analysis was performed using MEGA
software (version 1.01).
Salinity transfer experiment
Experimental protocol
1 week before experimentation, 46 fish were acclimated in each of 6 x
60 liter buckets supplied with 1 l min-1 Cypress creek water. Each
bucket was held in a large tank supplied with well water to maintain water
temperature at 8°C. Fish were not fed during the acclimation period or
during the salinity transfer experiment. 1 h before the salinity transfer (Pre
on graphs), four fish were removed from each tank and their tissues sampled
(see below). At time zero (vertical broken line on graphs), the water level in
each bucket was lowered to 12 liters and then immediately replaced with either
freshwater and/or seawater to yield salinity equivalent to 0%, 40% or 80%
seawater. Fish were held under static/renewal conditions where 75% of the
water was replaced daily. Salinity was monitored daily using a handheld
refractometer. Each transfer condition was duplicated and four fish were
sampled from each tank at 2, 4, 8, 24, 48, 72, 120, 240 and 360 h after
transfer, yielding a total of eight fish per treatment per time point. There
were no statistically significant differences for any measurements between
duplicate tanks, so all data were pooled and are presented as a single
point.
Tissue sampling
Individual fish were netted from each tank with minimal disturbance and
rapidly killed by concussion. Blood samples were taken by caudal puncture
using heparinized needles and stored briefly on ice. Plasma was separated from
blood cells by centrifugation and plasma was frozen in liquid N2.
Plasma samples were used for the measurement of osmolality, [Na+],
[Cl-] and [cortisol]. Immediately following blood sampling, second
and third gill arches were quickly dissected from each fish and frozen in
liquid N2.
Gill Na+/K+-ATPase enzyme activity
Gill Na+/K+-ATPase activity was measured on crude
gill homogenates using the methods outlined by McCormick
(1993). This assay couples
ouabain-sensitive ATP hydrolysis to the oxidation of NADH via
pyruvate kinase and lactate dehydrogenase. Briefly, gill filaments (
50
mg) were cut from each arch on ice and immediately homogenized in SEI buffer
(250 mmol l-1 sucrose, 10 mmol l-1
EDTA.Na2, 50 mmol l-1 imidazole, pH
7.3) with 0.1% sodium deoxycholate. Gill homogenates were centrifuged at 5000
g for 30 s and the supernatant immediately frozen in liquid
N2. To determine Na+/K+-ATPase activity,
homogenates were thawed and 10 µl was assayed for ATPase activity in the
absence or presence of 0.5 mmol l-1 ouabain. Each assay was run in
triplicate and the coefficient of variation was always <10%. Homogenate
[protein] was measured using the bicinchoninic acid method (Sigma-Aldrich)
with bovine serum albumin standards. Na+/K+-ATPase
activities were calculated by subtracting the ouabain sensitive ATPase
activity from total ATPase activity and are expressed in µmol
mg-1 protein h-1.
Plasma analysis
Plasma osmolality was measured using a Wescor 5500 vapour pressure
osmometer (Wescor Inc., UT, USA). Plasma [Na+] was measured using a
FLM2 flame photometer (Radiometer, Copenhagen, Denmark). Plasma
[Cl-] was measured using a CMT 10 chloride titrator (Radiometer).
When possible, all assays were done in duplicate. Plasma [cortisol] was
determined in duplicate using a [125I] radioimmunoassay (Medicorp
Inc., Montreal, QC, Canada). The coefficient of variation between duplicates
was <15%.
Gill Na+/K+-ATPase -isoform
expression
Na+/K+-ATPase -subunit isoform expression was
estimated using quantitative real-time PCR (qRT-PCR). Isoform-specific primers
were designed using Primer Express software (version 2.0.0; Applied Biosystems
Inc., Foster City, CA, USA). Primer sequences were as follows:
Na+/K+-ATPase
1a forward 5' GGC CGG CGA GTC
CAA T 3', Na+/K+-ATPase
1a reverse 5'
GAG CAG CTG TCC AGG ATC CT 3' (product size 66),
Na+/K+-ATPase
1b forward 5' CTG CTA CAT CTC
AAC CAA CAA CAT T 3', Na+/K+-ATPase
1b
reverse 5' CAC CAT CAC AGT GTT CAT TGG AT 3' (product size 81),
Na+/K+-ATPase
1c forward 5' GAG AGG GAG ACG
TAC TAC TAG AAA GCA 3', Na+/K+-ATPase
1c
reverse 5' CAG CAA GAC AAC CAT GCA AGA 3' (product size 69);
Na+/K+-ATPase
3 forward 5' CCA GGT ATT GAG
TTC CGT GTG A 3', Na+/K+-ATPase
3 reverse
5' CAG CCT GAA ATG GGT GTT CCT 3' (product size 66), and
elongation factor-1
forward 5' GAG ACC CAT TGA AAA GTT CGA GAA G
3', elongation factor-1
reverse 5' GCA CCC AGG CAT ACT TGA
AAG 3' (product size 71).
Total RNA was extracted from 20 mg of frozen gill tissue using TriPure
reagent and quantified spectrophotometrically. First-strand cDNA was
synthesized from 5 µg of total RNA using the protocols outlined above.
Quantitative RT-PCR was performed on an ABI Prism 7000 sequence analysis
system (Applied Biosystems Inc., Foster City, CA, USA). PCR reactions
contained 2 µl of cDNA, 4 pmoles of each primer and Universal SYBR green
master mix (Applied Biosystems Inc.) in a total volume of 20 µl. All
qRT-PCR reactions were performed as follows: 2 min at 50°C, 10 min at
95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min
Melt curve analysis was performed following each reaction to confirm the
presence of only a single product of the reaction. In addition, representative
PCR products were electrophoresed on a 2.0% agarose gel to verify that only a
single band was present. Negative control reactions were performed for all
samples using RNA that had not been reverse transcribed to control for the
possible presence of genomic DNA contamination. Genomic DNA contamination was
present in all samples, but never constituted more than 1:4096 starting copies
for Na+/K+-ATPase
1a, 1:16 384 starting copies
for Na+/K+-ATPase
1b, 1:2048 starting copies for
Na+/K+-ATPase
1c, 1:1024 starting copies for
Na+/K+-ATPase
3, or 1:16 384 starting copies for
the control gene elongation factor-1
(EF-1
; Accession No.
AF498320). As a result, only a negligible fraction of the qRT-PCR signal was
attributable to genomic DNA. One randomly selected control sample was used to
develop a standard curve relating threshold cycle to cDNA amount for each
primer set. All results were expressed relative to these standard curves and
mRNA amounts are normalized relative to EF-1
. Post-transfer samples are
expressed relative to the pre-transfer freshwater gill samples. All samples
were run in duplicate and the coefficient of variation between duplicate
samples was always <10%. To reduce cost, pre-transfer controls and 8 h, 5
day and 15 day post-transfer samples were analyzed first. If there were
differences in Na+/K+-ATPase
-isoform expression
at one of these time points, then the complete time course was analyzed;
however, if no differences in expression were observed then no further
analysis was performed.
The relative quantity of Na+/K+-ATPase-1a,
-
1b, -
1c and -
3, mRNA in trout gills was estimated for
each gene in the pre-transfer freshwater trout using the formula
EfficiencyCt, where efficiency is the slope of the standard
curve and Ct is the threshold cycle number. These quantities were also
expressed relative to EF-1
.
Statistical analysis
All data are presented as means ± S.E.M. (N,
number of fish). At each time point, values for trout held in 40% and 80%
seawater were compared to freshwater controls using a non-parametric
KruskalWallis test. Significance was set at =0.05, and when
obtained, Dunn's post-hoc test was used to identify where significant
differences occurred.
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Results |
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These five cDNA sequences have coding regions of 30333087
nucleotides, yielding an open reading frame of 10111029 aa
(Table 1). The five
Na+/K+-ATPase -isoforms were 72.689.0% and
74.689.2% similar at the cDNA (coding region) and aa levels,
respectively (Table 2). The
probable start codon (ATG) was between 21 and 159 nucleotides from the
5' end of the cDNA and the probable stop codon (TAT/TAC) was between 135
and 1440 nucleotides from the 3' poly(A)+ tail
(Table 1). Among the five
Na+/K+-ATPase
-isoforms, the 5'- and
3'-UTRs were only 1035% and 328% similar, respectively,
except for Na+/K+-ATPase
1a and
1b, which
shared 90% and 38% similarity in the 5' and 3' UTRs,
respectively.
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Sliding window analysis of the trout Na+/K+-ATPase
-isoforms identified three areas of low aa variability (<10%
heterogeneity) common to the five isoforms
(Fig. 2D). These areas of low
variability were located 320360, 580630 and 690755 aas
from the N terminus. Located within these areas of low variability were the
predicted ATP binding site (g in Fig.
2C), which was fully conserved in all trout isoforms, and a highly
conserved `hinge' sequence found in all P-type ATPases
(VAVTGDGVNDSPALRKADIGVAM; h in Fig.
2C) (Mobasheri et al.,
2000
). The only variation among isoforms in the `hinge' sequence
was a conservative aa substitution in the Na+/K+-ATPase
1a isoform. Two areas of high aa variability (>50% heterogeneity)
were found among the isoforms. The first area of high variability included the
40 N-terminal aas (ns; Fig.
2A,D) and the second was a 12-aa segment located approximately 490
aa from the N terminus (cs; Fig.
2B,D). Sliding window analysis of the
Na+/K+-ATPase
-isoform cDNA sequences identified
two areas of >75% variability (Fig.
2E), which correspond to the two areas of high variability
observed in the aa sequence (ns and cs;
Fig. 2C).
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Hydrophobicity analysis of each Na+/K+-ATPase
-isoform revealed nine putative transmembrane domains (f in
Fig. 2C), which is in good
agreement with the 810 transmembrane domains predicted to be present in
mammalian sequences (Mobasheri et al.,
2000
). One predicted transmembrane domain was located in an area
of low variability, while the other eight transmembrane domains were located
in areas of high variability (cf. Fig.
2C,D).
Each trout Na+/K+-ATPase -isoform had a
tissue-specific distribution. Na+/K+-ATPase
1c
and
3 isoforms were found in all tissues examined
(Fig. 3). Na+/K+-ATPase
1c was expressed in brain, eye,
gill, heart, kidney, spleen, intestine, liver, white muscle, red muscle and
testis. Na+/K+-ATPase
3 was ubiquitously
expressed. The Na+/K+-ATPase
1b was expressed in
brain, eye, gill, kidney, liver, spleen, intestine, testis, white muscle and
red muscle. The most restricted tissue distributions were observed for
Na+/K+-ATPase
1a and
2 isoforms
(Fig. 3).
Na+/K+-ATPase
1a isoform was found only in the
gills and heart of trout, while the
2 isoform was expressed in white
muscle, red muscle, brain and eye.
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Salinity transfer
Cortisol
Trout held in freshwater during the pre-transfer period (Pre) had plasma
[cortisol] of 1520 ng ml-1
(Fig. 4). Transfer of trout
from freshwater to freshwater (control; 0 salinity), 40% seawater
(14.4±0.11
; N=18), or 80% seawater
(27.8±0.09
; N=18) and subsequent holding for 15 days
did not significantly affect plasma [cortisol] at any time
(P>0.05).
|
Plasma ions
Transfer of trout from freshwater to 40% and 80% seawater resulted in a
rapid (2 h) and salinity-dependent increase in plasma osmolality, which
remained elevated compared to freshwater controls for the 15 day post-transfer
period (Fig. 5A). Similarly,
plasma [Na+] and [Cl-] increased in a salinity-dependent
manner following transfer from freshwater to 40% and 80% seawater
(Fig. 5B,C) and, for the most
part, remained elevated compared to the freshwater controls throughout the 15
day post-transfer period.
|
Gill Na+/K+-ATPase enzyme activity
Gill Na+/K+-ATPase activity did not change following
transfer from freshwater to either freshwater or 40% seawater
(Fig. 6). Furthermore, gill
Na+/K+-ATPase activity was unaffected during the first 5
days following transfer from freshwater to 80% seawater; however, compared to
the freshwater controls, gill Na+/K+-ATPase activity
increased 1.9- and 2.4-fold at 10 and 15 days post-transfer, respectively
(Fig. 6).
|
Na+/K+-ATPase -isoform expression
Of the four Na+/K+-ATPase -isoforms found in
freshwater trout gills,
1a was expressed at the highest level
(28.4±5.1-fold greater than EF-1
; N=8), followed by
1b,
1c and
3, which were expressed at 6.3±1.2,
0.13±0.03 and 0.000023±0.000009, respectively (expressed
relative to EF-1
). Following seawater transfer, two
Na+/K+-ATPase
-isoforms (
1a and
1b)
were differentially expressed in trout gills
(Fig. 7A,B), while the other
two isoforms (
1c and
3) were found not to respond to salinity
transfer (Tables 3 and
4). Furthermore, using qRT-PCR,
we verified that Na+/K+-ATPase
2 expression did
not change following seawater transfer (data not shown).
Na+/K+-ATPase
1a mRNA was expressed at high
levels in trout held in freshwater and dramatically decreased within 1 day
following transfer from freshwater to 40% and 80% seawater
(Fig. 7A). In contrast, trout
transferred from freshwater to 80% seawater experienced a transient increase
in Na+/K+-ATPase
1b mRNA compared to the
freshwater controls (Fig. 7B).
Transfer of trout from freshwater to 40% seawater did not affect gill
Na+/K+-ATPase
1b mRNA for the first 5 days
post-transfer, but significant decreases in gill
Na+/K+-ATPase
1b mRNA were observed at 10 and 15
days post-transfer (Fig.
7B).
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Discussion |
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Since the discovery of Na+/K+-ATPase -isoforms
in vertebrate tissues (Sweadner,
1979
), considerable effort has focused on elucidating the
biochemical differences among isoforms and the underlying molecular basis for
these differences (see Blanco and Mercer, 2001). Two areas of high variability
were present among the trout Na+/K+-ATPase
-isoforms. One area of high variability was located within the first 40
aa from the N terminus (Fig.
2A), and a second area of high variability was within a central 12
aa region starting approximately 490 aa from the N terminus
(Fig. 2B). The functional
significance of variation in the N-terminal and central isoform-specific
regions has not been fully characterized; however, there is consensus that
these regions may be involved in the regulation of
Na+/K+-ATPase activity by protein kinases C (PKC;
Efendiev et al., 2000
;
Pierre et al., 2002
). Within
the N-terminal region of rat Na+/K+-ATPase
1-isoform, the specific targets of PKC phosphorylation are Ser 16 and
Ser 23 (Efendiev et al., 2000
).
Trout Na+/K+-ATPase
1a-,
1b-,
1c-
and
3-isoforms all possess Ser 16, but only
1a and
1b
possess a serine close to position 23 (Ser 24). Furthermore, there are several
amino acids that differ among trout isoforms in the central isoform-specific
region. These sequence differences may allow for isoform-specific regulation
of Na+/K+-ATPase activity by PKC.
Further insight into the physiological roles of the trout
Na+/K+-ATPase -isoforms can be gained from their
tissue distribution (Mobasheri et al.,
2000
). In rats, the Na+/K+-ATPase
1-isoform is ubiquitously expressed, while
Na+/K+-ATPase
2 and
3 are expressed only
in muscle and excitable tissues, respectively
(Mobasheri et al., 2000
).
Na+/K+-ATPase
4 is found only in rat testis
(Woo et al., 2000
). Based upon
tissue distribution, the rat Na+/K+-ATPase
1-isoform is thought to be a `housekeeping' enzyme, while
Na+/K+-ATPase
2-,
3- and
4-isoforms
are thought to fulfil tissue-specific physiological functions. The tissue
distributions of trout Na+/K+-ATPase
1c and
2 are in good agreement with the tissue distribution patterns of rat
1 and
2, respectively. However, trout
Na+/K+-ATPase
1a and
1b show a restricted
tissue distribution and unlike the rat, trout
Na+/K+-ATPase
3 shows a ubiquitous tissue
distribution (Fig. 3) and no
4 isoform was identified in trout tissues
(Fig. 1). Therefore, by analogy
to the rat, trout Na+/K+-ATPase
1c and
3
probably function as `housekeeping' enzymes, while trout
Na+/K+-ATPase
1a,
1b and
2 are
likely to have specific physiological functions. For example,
Na+/K+-ATPase
2 is found predominately in muscle,
suggesting that this isoform may have the kinetic properties appropriate for
supporting excitation-contraction coupling.
Na+/K+-ATPase
1a and
1b are found in a
number of common tissues including gill and red muscle, but no precise
functional role can be ascribed from the tissue distribution
(Fig. 3).
During seawater transfer, many anadromous and euryhaline fish increase
their gill Na+/K+-ATPase enzyme activity to facilitate
ion secretion against a concentration gradient
(Marshall, 2002). These
increases in gill Na+/K+-ATPase activity are, in
general, either preceded by or accompanied by increases in
Na+/K+-ATPase
-subunit mRNA. Specifically,
increases in Na+/K+-ATPase
-subunit mRNA during
salinity transfer have been shown in Atlantic salmon Salmo salar
(D'Cotta et al., 2000
;
Singer et al., 2002
), brook
trout Salmo trutta (Madsen et
al., 1995
; Seidelin et al.,
2000
), the European eel (Cutler
et al., 1995a
), and the European sea bass Dicentrarchus
labrax (Jensen et al.,
1998
). Increases in Na+/K+-ATPase
1
and
3-subunit protein abundance also occur in tilapia gills following
seawater transfer (Lee et al.,
2003
,
1998
). However, there is
discordance among studies in the degree and timing of these changes in
Na+/K+-ATPase activity,
-subunit mRNA expression
and protein abundance. In addition, few studies have attempted to determine
whether Na+/K+-ATPase
-isoforms play a role in
facilitating freshwater or seawater acclimation.
The changes in plasma osmolality, [Na+], [Cl-]
(Fig. 5AC) and gill
Na+/K+-ATPase activity
(Fig. 6) following transfer to
80% seawater are in good agreement with previous studies in rainbow trout and
Coho salmon Oncorhynchus kisutch during seawater acclimation
(Fuentes et al., 1997;
Wilson et al., 2002
). Transfer
of trout from freshwater to 40% seawater had only minor affects on plasma
osmolality, [Na+] and [Cl-]
(Fig. 5AC) and did not
increase gill Na+/K+-ATPase activity
(Fig. 6). Trout held in 40%
seawater probably do not increase Na+/K+-ATPase activity
because 40% seawater is nearly isoosmotic with their blood and therefore does
not pose severe ionoregulatory stress to fish.
Of the four Na+/K+-ATPase -isoforms expressed
in trout gills (
1a,
1b,
1c and
3;
Fig. 3), two were
differentially regulated in response to seawater transfer. Expression of
Na+/K+-ATPase
1b mRNA increased in trout gills in
response to seawater transfer while the expression of
Na+/K+-ATPase
1a mRNA decreased in response to
seawater transfer. The present study is the first to demonstrate isoform
switching during seawater transfer and suggests that differential expression
of Na+/K+-ATPase
1a- and
1b-isoforms
during seawater transfer may be an important feature underlying the
transformation of the fish gill from an ion-absorbing epithelium to an
ion-secreting epithelium. These results, obtained using isoform-specific
qRT-PCR, are in contrast to all previous studies that showed a general
increase in Na+/K+-ATPase
-subunit mRNA following
seawater transfer, similar to the increase observed for
Na+/K+-ATPase
1b
(Fig. 7B). In fact, if we sum
our estimates for all Na+/K+-ATPase
-isoforms
monitored, we predict, based upon our quantitative estimates of
Na+/K+-ATPase
-isoform expression, that there
would be 3.5- and 2.2-fold decreases in total
Na+/K+-ATPase
-subunit expression (relative to
EF-1
) 3 days following transfer from freshwater to 40 and 80% seawater,
respectively. Similarly, at 15 days following transfer of trout from
freshwater to 40 and 80% seawater, we predict that total
Na+/K+-ATPase
-subunit expression would be 34-
and 17-fold lower than in the freshwater controls. Clearly, previous studies
using northern blot analysis were biased toward monitoring a
Na+/K+-ATPase
1b-like isoform in fish gills
during seawater transfer and thus do not provide a complete picture of the
role Na+/K+-ATPase in freshwater and seawater
acclimation.
Despite decreases in total Na+/K+-ATPase
-isoform mRNA in trout gills following transfer from freshwater to 80%
seawater, there was a 2.4-fold increase in gill
Na+/K+-ATPase activity at 15 days
(Fig. 6). The increase in gill
Na+/K+-ATPase activity following transfer to 80%
seawater was probably initiated by the transient increase in
Na+/K+-ATPase
1b mRNA expression
(Fig. 7A) and sustained by
translational controls. Furthermore, post-translational modification of
Na+/K+-ATPase (e.g. Na+/K+-ATPase
-subunit binding; Béguin et
al., 1997
; Therien et al.,
1999
) may also contribute to the sustained elevation of
Na+/K+-ATPase activity following seawater transfer.
Na+/K+-ATPase 1a is expressed at high levels
in the freshwater fish gill and decreases within 1 day following transfer from
freshwater to 40% and 80% seawater. This downregulation of
Na+/K+-ATPase
1a during seawater transfer
suggests that this isoform may have kinetic properties favourable for
ionregulation in freshwater. Cation binding affinity to
Na+/K+-ATPase is modulated by differences in the amino
acid residues present in the 4th, 5th and 6th
transmembrane domains of the
-subunit
(Mobasheri et al., 2000
).
Among the trout Na+/K+-ATPase
-isoforms, the
amino acid sequences of the 5th and 6th transmembrane
domains are highly variable, while the amino acid sequence of the
4th transmembrane is well conserved. For the most part, the
differences between trout Na+/K+-ATPase
-isoforms
tend to separate Na+/K+-ATPase
1a from the other
four isoforms. In particular, within the 5th predicted
transmembrane domain (Fig. 2B),
7 out of 21 amino acids differ in Na+/K+-ATPase
1a compared to the other four isoforms. It is tempting to speculate
that these differences in amino acid sequence in
Na+/K+-ATPase
1a may alter cation binding
affinity and facilitate Na+ and Cl- uptake from
freshwater.
Gene expression in fish gills during seawater transfer is thought to be
primarily mediated by changes in circulating hormones
(McCormick, 1995). Following
seawater transfer, many studies have reported increases in plasma [cortisol]
(Marshall et al., 1999
;
McCormick, 2001
;
Wilson et al., 2002
), which
has been implicated in initiating changes in gene expression. For example, a
large and rapid (1 h) increase in plasma [cortisol] was found to precede an
increase in cystic fibrosis transmembranse conductance regulator (CFTR)
expression in Fundulus heteroclitus gills following seawater transfer
(Marshall et al., 1999
;
Singer et al., 1998
). In the
present study, seawater transfer did not significantly affect plasma
[cortisol]; however, at 1 day post-transfer, there was a highly variable,
salinity-dependent increase in plasma [cortisol]. This variable increase in
plasma [cortisol] agrees well with a secondary spike in [cortisol] observed by
Marshall et al. (1999
) and
coincides with changes in Na+/K+-ATPase
1a and
1b mRNA in trout gills following seawater transfer (c.f. Figs
4 and
7A,B). Therefore, it is
possible that changes in plasma cortisol may be involved in the regulation
Na+/K+-ATPase
1a and
1b transcription in
trout. It should be noted, however, that additional hormones are known to
change in response to salinity transfer (e.g. insulin-like growth factor,
growth hormone; McCormick,
1996
), and may influence Na+/K+-ATPase gene
expression.
Changes in gill morphology during salinity transfer may also influence the
pattern of Na+/K+-ATPase gene expression. Trout gills
are composed of multiple cell types including respiratory pavement cells and
ion-regulating mitochondria-rich (MR; chloride) cells. Recently, two distinct
populations of MR cells have been isolated from trout gills
(Galvez et al., 2002;
Goss et al., 2001
) and there
is accumulating evidence that these two MR cell populations may differ in
their complement of ion transporters; thus it seems likely that these two cell
populations may be differentially proliferated during freshwater and seawater
acclimation. Differential changes in cell proliferation during salinity
transfer may explain some of the observed changes in
Na+/K+-ATPase
-isoform expression
(Fig. 7A,B). For example, if
Na+/K+-ATPase
1a is expressed in only the MR-cell
type that proliferates in freshwater and decreases in number or size during
seawater transfer, then decreases in whole gill
Na+/K+-ATPase
1a mRNA may reflect changes in cell
proliferation and not changes in global gene expression. Future research
should examine the relationship between Na+/K+-ATPase
1-isoform expression and MR-cell population during salinity
transfer.
In conclusion, in the present study we identified five
Na+/K+-ATPase -isoforms in rainbow trout and
showed that each isoform had a tissue specific distribution pattern. Two of
the Na+/K+-ATPase
-isoforms (
1a and
1b) were differentially expressed in gills during transfer from
freshwater to 80% seawater. Expression of Na+/K+-ATPase
1b was upregulated in response to seawater acclimation suggesting a
role in ion secretion, while expression of Na+/K+-ATPase
1a was downregulated in response to seawater acclimation, suggesting a
role in ion uptake from freshwater. Clearly,
Na+/K+-ATPase
-isoform switching during seawater
transfer provides new insight into the importance of this gene in fish ion
regulation.
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Acknowledgments |
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Footnotes |
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References |
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