Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport
1 Department of Zoology, University of British Columbia, Vancouver BC,
Canada V6T 1Z4
2 Department of Biology, Georgia Southern University, Statesboro, GA
30460-8042, USA
3 Department of Physiology and Pharmacology, James Cook University, Cairns,
QLD 4879, Australia
4 Department of Biology, McMaster University, Hamilton ON, Canada L8S
4K1
* Author for correspondence (e-mail: scott{at}zoology.ubc.ca)
Accepted 17 May 2005
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Summary |
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Key words: Fundulus heteroclitus, carbonic anhydrase, Na+, H+-exchanger, Na+, HCO3cotransporter, fish
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Introduction |
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In addition to many studies on killifish in vivo, an early finding
of great importance to understanding branchial ion transport was the discovery
that skin lining the opercular bone of this species was similar to the gills
with regards to many morphological and functional characteristics
(Burns and Copeland, 1950;
Karnaky et al., 1977
). The
flat opercular epithelium has since been the subject of numerous studies, many
of which have established the mechanisms of ion secretion in seawater teleosts
(Wood and Marshall, 1994
;
Marshall and Bryson, 1998
).
Although this preparation appears to be an excellent surrogate model for
understanding the physiology of gills in seawater fish, recent findings
suggest that the opercular epithelium is not an adequate model for gill
physiology in freshwater fish. Briefly, several studies of whole animal ion
flux have demonstrated that intact killifish in vivo actively absorb
Na+ at high rates, while Cl uptake is passive and
negligible in freshwater (Patrick et al.,
1997
; Patrick and Wood,
1999
; Wood and Laurent,
2003
). In contrast, when the opercular epithelium of
freshwater-acclimated killifish is mounted in vitro with freshwater
on the apical surface, it absorbs Cl actively at low rates
and Na+ uptake is passive
(Marshall et al., 1997
;
Burgess et al., 1998
). Only 1%
of Na+ and Cl transport in the intact animal is
accomplished by the opercular epithelium
(Degnan and Zadunaisky, 1979
),
so the patterns of ion flux in killifish in vivo are likely
representative of what occurs across the gills. Although the molecular
mechanisms of Na+ absorption across killifish gills are poorly
understood, apical Na+,H+-exchanger (NHE), basolateral
Na+,K+-ATPase, and possibly V-type H+-ATPase
(V-ATPase) are believed to be important
(Patrick et al., 1997
;
Patrick and Wood, 1999
;
Katoh et al., 2003
;
Edwards et al., 2005
). Some
other genes thought to be important in freshwater fish gills, such as carbonic
anhydrase (CA) and
Na+,HCO3cotransporter (NBC), may also
be involved (Wood and Pärt,
2000
; Marshall,
2002
; Perry et al.,
2003a
,b
).
The mechanisms of Cl absorption across the opercular
epithelium, and why these mechanisms are absent from the gills, are largely
unknown (see Marshall et al.,
1997
; Burgess et al.,
1998
).
While its similarity to the gills has made the opercular epithelium useful
for understanding some aspects of fish gill physiology, the functional
differences between these tissues in freshwater provide an excellent
comparative model to study the molecular basis of physiological function. In
particular, differences in gene expression between gills and opercular
epithelium could be responsible for their differences in Na+ and
Cl transport in freshwater. An important objective of the
present study was therefore to compare the expression of several ion transport
genes in gills and opercular epithelium of killifish. Because little is known
about the role and expression of some of the genes cloned in the present study
(e.g. NHE2, NHE3, CA2 and NBC1), an additional objective was to characterize
the expression patterns of these genes after abrupt transfer from brackish
water (10% seawater) to freshwater. Near-isosmotic brackish water is the
preferred salinity for F. heteroclitus
(Fritz and Garside, 1974), and
transfer from brackish water to freshwater may be more environmentally
representative of the conditions that killifish naturally encounter in
estuaries
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Materials and methods |
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Total RNA extraction, reverse transcription and cloning
Genes of interest were cloned from killifish gills, which were sampled as
described below. Total RNA was extracted using either Trizol (Invitrogen,
Burlington, ON, Canada) or Tri-Reagent (Sigma-Aldrich, Oakville, ON, Canada)
isolation reagents following the manufacturer's instructions. RNA
concentrations were determined spectrophotometrically and RNA integrity was
verified by agarose gel electrophoresis [1% (w/v)
agarose:Tris-acetate-EDTA]. Extracted RNA samples were stored at
80°C following isolation. First strand cDNA was synthesized by
reverse transcribing total RNA using oligo(dT) primer and either RevertAid H
Minus M-MuLV reverse transcriptase (MBI Fermentas, Burlington, ON, Canada) or
Superscript II RNAse H-reverse transcriptase (Invitrogen), following each
manufacturer's protocols.
Several genes of potential importance for ion regulation in fish were
cloned using a polymerase chain reaction (PCR)-based approach. Multiple
alignments of previously published cDNA sequences were constructed using
ClustalW (Thompson et al.,
1994) to identify conserved gene regions, from which primers were
then designed (Table 1).
Specific sequences within CA2, NBC1, NHE2, and NHE3 were amplified from
killifish gills using Taq polymerase (MBI Fermentas or Invitrogen).
Each PCR consisted of 3540 cycles of 3060 s at 94°C,
3060 s at the lower annealing temperature for each respective primer
set (see Table 1), and 60 s for
every 1000 bp of expected product at 72°C. PCR products were verified by
electrophoresis on 1% agarose gels containing ethidium bromide and cloned into
pGEM-T Easy (Promega, Nepean, ON, Canada) or pCR2.1 (Invitrogen) vector
plasmid. Multiple clones of each fragment were sequenced bidirectionally, and
the partial CA2, NBC1 and NHE3 consensus sequences obtained were submitted to
the GenBank database (CA2, accession no. AY796057; NBC1, acc. no. AY796058;
NHE3, acc. no. AY818825).
|
The complete cDNA sequence of NHE2 was subsequently obtained by rapid amplification of cDNA ends (RACE). Initial PCR amplification of the 3' region was achieved using a killifish gene-specific primer (5'-TCT TTG TGG GAC TGT TCT TCG GCT TG-3') and the Invitrogen GeneRacer 3' primer. Similarly, amplification of the 5' region used a gene-specific primer (5'-GGT CTC ACT CAC GCT ACT CCA CAT C-3') and the Invitrogen GeneRacer 5' primer. For both 3' and 5' RACE protocols, the possibility of PCR artifact was reduced by using nested RACE-PCR, which used 1 µl of the original amplification reaction as a template, killifish gene specific primer (3' RACE: 5'-TCC CCT CTT CGT CTT CCT CTA CTC-3' ; 5' RACE: 5'-GCT CTG ACA CAT TCG CTT CCA C-3'), and the GeneRacer 3' or 5' nested primer. Thermal cycling parameters for all reactions included an initial incubation at 95°C (5 min), followed by 35 cycles of 94°C (30 s), 60°C (90 s), and 72°C (180 s), and a final extension at 72°C (7 min). The resulting products were visualized, sub-cloned, and sequenced as previously described, and the complete NHE2 consensus sequence was submitted to GenBank (NHE2, acc. no. AY818824).
In addition to the sequences cloned in this study, several other killifish
cDNA sequences were used in this work for real-time PCR analysis of gene
expression. These sequences were for the signalling protein 14-3-3a (acc. no.
AF302039), cystic fibrosis transmembrane conductance regulator
Cl channel (CFTR; acc. no. AF000271), elongation factor
1 (EF1
; acc. no. AY430091), Na+,K+-ATPase
1a (acc. no. AY057072),
Na+,K+,2Clcotransporter 1 (NKCC1; acc.
no. AY533706), and V-type H+-ATPase subunit A (V-ATPase; acc. no.
AB066243).
Salinity transfer protocol
Fish were acclimated to a salinity of 10% seawater (brackish water) for at
least 1 month before salinity transfer. In experiment 1,
Na+,K+-ATPase activity was measured in the gills and
opercular epithelia of fish before transfer, or 12 h, 3 days or 7 days after
transfer to freshwater. In experiment 2, mRNA expression in the gills and
opercular epithelia was measured 12 h, 3 days or 7 days after transfer either
to brackish water (i.e. a sham treatment where the animals were simply
transferred between two tanks of their same acclimation salinity) or to
freshwater. In all cases, fish were transferred between tanks with a net. Fish
were sampled by cephalic blow followed by rapid decapitation, second and third
whole gill arches were removed, opercular epithelia were isolated by scraping
off the underside of the opercular bone, and both tissues were immediately
frozen in liquid nitrogen. Tissues were not perfused before freezing because
blood has been previously shown to contribute little to whole-gill expression
levels (Perry et al., 2000;
Scott et al., 2004a
). All
tissues were stored at 80°C until analyzed.
Na+,K+-ATPase activity assay
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
(1993). For this assay, gills
and opercular epithelia were homogenized in 500 µl of SEI buffer (150 mmol
l1 sucrose, 10 mmol l1 EDTA, 50 mmol
l1 imidazole, pH 7.3) containing 0.1% sodium deoxycholate
and centrifuged at 5000 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
mmol l1 ouabain using 10 µl 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
µmol ADP mg1 protein h1.
Real-time PCR analysis of gene expression
Total RNA was extracted and reverse transcribed from killifish gills using
the methods described above. Gene expression was assessed using quantitative
real-time PCR (qRT-PCR) on an ABI Prism 7000 sequence analysis system (Applied
Biosystems, Foster City, CA, USA). 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 pmoles 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. Whereas some
genes were cloned using killifish from populations in Georgia and Maine, the
fish used in the freshwater transfer experiment were from Nova Scotia
(described above). All primers used for qRT-PCR were therefore tested in
multiple killifish populations, and they were used only if the melt-curve
analysis indicated that the same product was amplified in all populations.
Control reactions were conducted with no cDNA template or with
non-reverse-transcribed RNA to determine the level of background and genomic
DNA contamination, respectively. Genomic contamination was always below 1:35
starting cDNA copies for all templates, with the exception of NHE2 in
opercular epithelium (NHE2 could not be amplified above background levels in
opercular epithelium).
|
A randomly selected sample was used to develop a standard curve for each
primer set, and all results were expressed relative to this arbitrary
standard. All samples were run in duplicate (coefficients of variation were
10%). Results were then normalized to EF1
. Expression of this gene
does not change in killifish gills at any time following salinity transfer
when expression is normalized to total RNA concentration (data not shown),
demonstrating that EF1
is an appropriate control gene (see also
Richards et al., 2003
;
Scott et al., 2004a
). For
clarity in graphing, time course mRNA expression data are expressed relative
to the 12 h brackish water control samples. In addition, the absolute level of
mRNA expression of each gene examined was estimated semi-quantitatively 12 h
after transfer using the following formula:
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Statistical analyses
Data are expressed as means ±
S.E.M. All data passed tests of normality and
homogeneity of variance. Analysis of variance (ANOVA) was therefore used to
ascertain overall differences as a function of time for each salinity, as well
as overall differences in absolute expression levels for each tissue. Because
measured variables may change due to handling of fish alone
(Scott et al., 2004a), the
effect of freshwater transfer was assessed whenever possible by comparison
with time-matched brackish water controls using Tukey post-hoc
comparisons. The effect of handling was assessed using Tukey comparisons with
12 h brackish water controls. When time-matched controls were not available
(Na+,K+-ATPase activity experiment) the effect of
freshwater transfer was assessed by comparison with pre-transfer controls.
Relative expression levels of genes were compared between tissues using Tukey
post-hoc comparisons. All statistical analyses were conducted using
Sigmastat version 3.0 and a significance level of P<0.05 was used
throughout.
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Results |
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Gene expression after freshwater transfer
Freshwater transfer changed the mRNA expression patterns of some genes in
similar ways in gills and opercular epithelium of killifish. Expression of the
important seawater ion transporters NKCC1 and CFTR decreased in the gills and
opercular epithelium at all times after freshwater transfer, reaching levels
as low as 30% and 10% of brackish water controls
(Fig. 3). The expression of
14-3-3a, which is an important gene for regulating ion secretion, increased up
to 1.5-fold at 12 h and 7 days after freshwater transfer, but was similar to
brackish water controls at 3 days (Fig.
3). The mRNA expression of some other genes was unchanged by
freshwater transfer in both tissues, including
Na+,K+-ATPase 1a, NBC1 and V-ATPase
(Table 3).
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|
For some other genes, the patterns of expression exhibited in each tissue differed after freshwater transfer. In the gills, mRNA expression of NHE2 and CA2 increased in freshwater: NHE2 increased transiently at 12 h (1.7-fold) (Fig. 4), while CA2 increased up to twofold above controls at all times after transfer (Fig. 5). In contrast, NHE2 was not detectable in opercular epithelium and CA2 expression decreased in freshwater (Fig. 5). Curiously, NHE3 expression also differed between tissues, in a manner opposite the patterns exhibited by CA2, as expression of this gene decreased in the gills but increased in the opercular epithelium after freshwater transfer (Fig. 5). In a few cases, changes occurred over time in the brackish water controls in gills (14-3-3a and CA2) and opercular epithelium (NBC1), and this was likely an effect of handling.
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Discussion |
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Suppression of ion secretion after freshwater transfer
The opercular epithelium has proved a useful surrogate model for the gills
in seawater fish, and previous studies have shown that their mechanisms of ion
secretion are likely similar (Wood and
Marshall, 1994; Wood and
Laurent, 2003
). In this regard our study is no exception, as the
absolute expression levels of many genes involved in ion secretion
(Na+,K+-ATPase
1a, NKCC1 and CFTR)
were similar between gills and opercular epithelium, as well as the signaling
protein 14-3-3a, which is thought to regulate the activity of CFTR
(Kohn et al., 2003
).
In addition to its use as a model for ion secretion in seawater, the
opercular epithelium of killifish has also been useful for understanding how
ion secretion is suppressed in hyposmotic environments
(Marshall, 2003). Indeed, our
results suggest that the gills and opercular epithelium may behave similarly
in this regard, as the responses of seawater ion transporters to freshwater
transfer in these tissues were similar. Expression of NKCC1 and CFTR decreased
after freshwater transfer in both gills and opercular epithelium, and the
patterns of these changes were similar in each tissue. These results are also
consistent with previous studies of freshwater transfer for NKCC1 and CFTR
mRNA levels in the gills of killifish (Scott et al.,
2004a
,b
),
as well as NKCC protein abundance in the gills of other species
(Tipsmark et al., 2002
;
Wilson et al., 2004
).
Furthermore, genes expressed by the opercular epithelium (and possibly the
gills as well) also undergo many post-transcriptional alterations after
freshwater transfer to suppress ion secretion, including transporter
internalization and inactivation (Marshall et al.,
1998
,
2000
,
2002
;
Hoffmann et al., 2002
) and
morphological restructuring (Daborn et al.,
2001
). These post-transcriptional events appear particularly
important during short-term salinity change
(Marshall, 2003
).
The signaling protein 14-3-3a is potentially important for inactivating
Cl secretion via CFTR after freshwater transfer.
Previous studies have shown that killifish 14-3-3a promotes ionic homeostasis
in Xenopus oocytes (Kohn et al.,
2003), and mRNA expression of this gene increases in the gills of
killifish transferred to freshwater
(Kültz et al., 2001
). In
this study, 14-3-3a expression increased in both gills and opercular
epithelium after freshwater transfer, suggesting that some of the
intracellular signals responsible for inactivating ion secretion are similar
in each tissue. However, the intracellular signalling pathways that transduce
osmotic signals (see Kültz and Avila,
2001
; Seale et al.,
2003
; Marshall et al.,
2005
) are still unclear, so the direct role of 14-3-3a remains
uncertain.
Mechanisms of ion absorption
Although the absolute mRNA expression levels of many genes are similar
between gills and opercular epithelium, some other genes appear to be
expressed at substantially higher levels in the gills. Interestingly, those
genes whose expression differs between tissues, namely
Na+,H+-exchanger 2,
Na+,HCO3cotransporter 1, carbonic
anhydrase 2, and V-type H+-ATPase A, have all been proposed to be
important in the gills of various fish species for Na+ uptake in
freshwater (Claiborne et al.,
2002; Katoh et al.,
2003
; Perry et al.,
2003a
,b
).
With few exceptions, most previous studies in fish have found that changes in
the mRNA expression of ion transporters occur along with predictable changes
in protein abundance (e.g. D'Cotta et al.,
2000
; Tipsmark et al.,
2002
; Scott et al.,
2004a
; Scott et al.,
2005
). Assuming that the relative levels of transcription measured
in this study are correlated with levels of protein expression, it is
therefore plausible that the differences in ion transport between gills and
opercular epithelium are a result of the observed differences in absolute gene
expression: gills express higher levels of NHE2, NBC1, CA2, and V-ATPase and
are thus able to actively absorb Na+.
Based on the molecular and physiological differences between gills and
opercular epithelium, we have proposed a preliminary model on which to base
future investigation of how Na+ absorption might be accomplished
across the killifish gill epithelium (Fig.
6). In the model, apical Na+ uptake occurs via
NHE2 in exchange for H+ supplied by CA2. Active transport of
Na+ across the basolateral surface is accomplished by
Na+,K+-ATPase (which creates the electrochemical
gradients necessary for transport); therefore, apical Na+ uptake is
not driven by a transepithelial H+ gradient as in some other fish
species (Lin and Randall,
1993; Fenwick et al.,
1999
). Support for these suggestions come from immunological
evidence that CA2 and Na+,K+-ATPase localize to
mitochondria-rich cells in killifish gills
(Flügel et al., 1991
;
Katoh et al., 2003
). Our model
assumes a co-localization of NHE2 and CA2, and although CA2 is known to be
located at the apical surface (Rahim et
al., 1988
), the localization of NHE2 in killifish gills has yet to
be demonstrated.
|
In addition to NHE2, CA2 and Na+,K+-ATPase, NBC1 and
V-ATPase may be important for Na+ uptake and/or regulating
acidbase balance in freshwater. Na+ may cross the
basolateral surface in symport with HCO3 through
NBC1, which can operate in both influx
(1Na+:2HCO3;
Wood and Pärt, 2000;
Perry et al., 2003a
) and
efflux (1Na+:3HCO3, as shown in
Fig. 6; Hirata et al., 2003
) modes in
freshwater. NBC1 has not yet been localized in killifish chloride cells, but
V-ATPase is basolaterally located in killifish gill cells
(Katoh et al., 2003
), and
could therefore export protons across the basolateral surface. Each of these
proteins are likely involved in maintaining acidbase balance in
freshwater (e.g. Perry et al.,
2003a
), but may also support Na+ absorption. For
example, the absolute expression of V-ATPase was low in the moderately hard
freshwater used in this study, where environmental Na+ and
Cl concentrations were similar. However, basolateral
localization of this protein in killifish gills increased substantially in an
artificial freshwater medium with low Na+ content (12% of present
values) but high Cl (130% of present values), an ionic
situation that would tend to promote acidosis
(Katoh et al., 2003
). In this
situation, increased basolateral V-ATPase would not be beneficial for
regulating acidbase balance (as it would promote internal acidosis),
and may instead perform a different function. It has been suggested that
basolateral H+-ATPase facilitates transepithelial
Cl uptake in the gills of some fish species by extruding
protons, and thus driving apical
Cl,HCO3 exchange to maintain
intracellular acidbase balance
(Piermarini and Evans, 2001
;
Piermarini et al., 2002
).
However, killifish gills appear to lack apical
Cl,HCO3 exchange and do not
absorb Cl in freshwater
(Patrick et al., 1997
;
Patrick and Wood, 1999
;
Wood and Laurent, 2003
;
Scott et al., 2004b
), so
basolateral proton extrusion might instead be involved in Na+
uptake or other physiological functions that are necessary in dilute
environments. The exact roles of V-ATPase and NBC1 in Na+ uptake,
their stoichiometry relative to other transporters, and whether these proteins
are expressed in the same cell type as NHE2 and CA2, have yet to be
determined.
It is possible that some of the differences in expression between gills and
opercular epithelium result from differences in cell type composition within
each tissue. The contribution of different cell types (including cells not
involved in transport, such as connective tissue) to whole-tissue expression
cannot be resolved in this study. However, we hypothesize that the majority of
the observed differences seen in this study are due to mRNA expression in the
cells responsible for transepithelial ion transport. Previous studies have
demonstrated that ion transporters tend to be expressed at much higher levels
in these cells (e.g. Wilson et al.,
2000; Piermarini and Evans,
2001
), so their contribution to whole-tissue expression is large.
For the genes thought to be involved in ion secretion (NKCC1,
Na+,K+-ATPase
1a, CFTR and 14-3-3a),
as well as for NHE3, absolute expression levels in this study were the same in
the two tissues. Furthermore, mRNA expression of both NKCC1 and CFTR were
reduced after freshwater transfer, and this reduction was of a similar
magnitude in both tissues. Therefore, at least for these genes, there is no
apparent evidence that differences in cell type composition (either due to
non-epithelial cells, or differences in the relative abundance of
mitochondria-rich cells or pavement cells) are causing differences in
expression. Regardless, the contribution of cell type composition in
expression studies using whole-tissue deserves future attention.
The mechanisms through which the opercular epithelium of killifish actively
absorbs Cl, albeit at a low rate, are presently unclear
(Wood and Marshall, 1994;
Marshall et al., 1997
;
Burgess et al., 1998
), and
remain so because the only differences in expression that we detected between
tissues were of genes that likely function in Na+ absorption.
Cl uptake is unaffected by mucosal exposure to the anion
exchanger inhibitors SITS
(4-acetamino-4'-isothiocyanostilbene-2,2'-disulfonic acid) and
DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)
(Marshall et al., 1997
),
suggesting that transport may not occur by apical
Cl,HCO3 exchange.
Interestingly, CFTR appears to localize to the basolateral surface of pavement
cells in killifish opercular epithelium in freshwater, while it is not
expressed in this cell type in seawater
(Marshall et al., 2002
). This
transporter may therefore facilitate Cl transport by
pavement cells in freshwater. However, CFTR expression decreases substantially
after freshwater transfer in opercular epithelium, so if pavement cells
transport Cl via CFTR this may explain the
detectable but low rate of Cl uptake by the opercular
epithelium in vitro. Future work should address the role of CFTR and
other Cl transporters (e.g. SLC26 anion exchangers or
ClATPase; Dópido
et al., 2004
) in the opercular epithelium of killifish in
freshwater.
Activation of Na+ absorption after freshwater transfer
Killifish rapidly activate Na+ absorption after freshwater
transfer (Wood and Laurent,
2003; Scott et al.,
2004a
), and this occurs through many coordinated molecular and
cellular events. These are likely to include rapid activation of
Na+,K+-ATPase activity
(Towle et al., 1977
;
Mancera and McCormick, 2000
),
increased cell proliferation (Katoh and
Kaneko, 2003
; Scott et al.,
2005
), cell differentiation and apoptosis
(Marshall et al., 1999
;
Daborn et al., 2001
;
Katoh et al., 2001
) and, in
some cases, increased transcription of Na+,K+-ATPase
(Scott et al.,
2004a
,b
).
Expression of both Na+,H+-exchanger 2 and carbonic
anhydrase 2 mRNA increased as early as 12 h after transfer, so transcriptional
regulation of these genes may also play a role in the early stages of
freshwater acclimation. Changes in expression after salinity change can occur
within a few hours of transfer (Scott et
al., 2004a
), so NHE2 and CA2 expression may have increased earlier
than 12 h into freshwater. Some evidence suggests that changes in whole-gill
expression of ion transporters after freshwater transfer may be partly due to
alterations in cell proliferation (Scott
et al., 2005
). If this is indeed the case, differences in cell
proliferation and/or regression between gills and opercular epithelium would
be expected after freshwater transfer.
The expression of Na+,H+-exchanger proteins has been
reported in the gills of numerous fish species in freshwater
(Wilson et al., 2000;
Edwards et al., 2002
;
Hirata et al., 2003
) and their
expression is known to be modulated by acidbase disturbances
(Claiborne et al., 1999
;
Edwards et al., 2001
). Few
studies have examined how these proteins are modulated by freshwater transfer
(Claiborne et al., 1999
), but
it is reasonable to propose that NHE2 upregulation might contribute to
Na+ uptake in killifish. Although the role of carbonic anhydrase in
ion absorption is well established
(Marshall, 2002
;
Perry et al., 2003b
) and CA2
upregulation is probably also involved in activating Na+ uptake
after freshwater transfer, few previous studies have assessed its mRNA
expression patterns after transfer to hyposmotic environments
(Henry et al., 2003
). The
suppression of CA2 expression after transfer in opercular epithelium, a tissue
that is unable to actively absorb Na+, is consistent with a role
for this gene in Na+ uptake.
Previous studies in killifish observed Na+,K+-ATPase
1a mRNA expression and activity to increase after freshwater
transfer (Scott et al.,
2004a
,b
;
Scott et al., 2005
). The lack
of similarity in expression patterns between this and previous studies was
likely due to differences in experimental protocols: killifish were
transferred from 10% seawater to freshwater ([Na+] 0.82 mmol
l1) in this study, while in previous studies fish were
transferred from 30% seawater to freshwater with appreciably lower ion levels
([Na+] 0.17 mmol l1). The latter case represents
a much greater change in ionic gradients after transfer than in the present
study, and illustrates an important characteristic of physiological responses
to environmental change. That is, the presence and magnitude of the
physiological response is dependent on the degree of change, and in many cases
not all elements of a physiological system need to be adjusted to maintain
homeostasis. It is therefore likely that the post-transcriptional increase in
gill Na+,K+-ATPase activity in this study (which has
also been observed in two other studies; see
Towle et al., 1977
;
Mancera and McCormick, 2000
)
sufficiently activated Na+ transport to maintain ion balance.
Unlike the gills, the opercular epithelium did not increase Na+,K+-ATPase activity after freshwater transfer. This suggests that while the signalling pathways responsible for eliminating ion secretion after freshwater transfer may be similar between these tissues (e.g. 14-3-3a pathway), those activating ion absorption may be different. Furthermore, increasing Na+,K+-ATPase activity likely plays a lesser role in increasing Cl uptake in the opercular epithelium after freshwater transfer than for increasing Na+ uptake in the gills. Because Na+,K+-ATPase is important for ion secretion by both gills and opercular epithelium in seawater, however, it is perhaps not surprising that the absolute mRNA expression of this gene is the same in each tissue. The difference between these tissues may instead be in the post-transcriptional regulation of Na+,K+-ATPase activity.
In contrast to the expression of NHE2, which increased after freshwater
transfer, the expression of NHE3 decreased in the gills of killifish after
freshwater transfer, suggesting that there are freshwater- and
seawater-specific isoforms responsible for
Na+,H+-exchange (see also
Edwards et al., 2005). There
may be functional differences between these isoforms in kinetics, substrate
affinity, regulation or other properties that enabled selection for isoform
switching between different osmotic environments. In this regard, isoform
switching appears to be an important characteristic of the acclimatory
responses of fish to salinity change
(Schulte, 2004
). In salmonids,
for example, Na+,K+-ATPase
-isoform switching
occurs after transfer from freshwater to seawater
(Richards et al., 2003
).
Curiously, NHE3 expression increased in the opercular epithelium after
freshwater transfer, which further demonstrates the divergent mechanisms of
ion transport between these tissues in hyposmotic environments.
Taken together, the results of this study demonstrate the potential molecular basis for the differences in function between the gills and opercular epithelium of killifish in freshwater. While these tissues appear to behave similarly in seawater, at both the molecular and physiological levels, and while they also appear to suppress ion secretion in hyposmotic environments using similar mechanisms, their functional and genomic responses to freshwater transfer are markedly different. The gills of killifish actively absorb Na+ in freshwater while opercular epithelia do not, and this is likely based on substantial differences in the absolute expression of several proteins important for Na+ transport, namely NHE2, CA2, NBC1 and V-ATPase. Furthermore, the physiological responses of the gills during acclimation to freshwater occur in conjunction with augmented mRNA expression of NHE2 and CA2, events that did not occur in the opercular epithelia. Our results provide insight into the molecular and physiological mechanisms underlying ion transport in killifish gills, and demonstrate the substantial functional plasticity of ion transport in this species.
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