Induction of branchial ion transporter mRNA expression during acclimation to salinity change in the euryhaline crab Chasmagnathus granulatus
1 Departamento de Biodiversidad y Biología Experimental, Facultad de
Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pab. II, Ciudad
Universitaria, C1428EHA Buenos Aires, Argentina
2 CONICET (Consejo Nacional de Investigaciones Cientificas y Tecnicas),
Rivadavia 1917, C1033AAJ Buenos Aires, Argentina
3 Department of Animal Physiology, University of Osnabrueck, 49076
Osnabrueck, Germany
4 College of the Atlantic, Bar Harbor, ME 04609, USA
5 Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672,
USA
Author for correspondence (e-mail:
dtowle{at}mdibl.org)
Accepted 1 August 2005
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Summary |
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Key words: Na+/K+-ATPase, Na+/K+/2Cl- cotransporter, V-type H+-ATPase, arginine kinase, crab, gill, gene expression, quantitative PCR
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Introduction |
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Ultrastructural studies of C. granulatus gills show that a
morphology characteristic of ion-transporting cells predominates in posterior
gills (Luquet et al., 2000).
Within 25 days following transfer from seawater to 12
salinity, the
depth of septate junctions between epithelial cells in posterior gills
increased significantly, suggesting that the gill epithelium is less permeable
in reduced salinity (Luquet et al.,
2002a
). In addition, the abundance of the ion-transporting cell
type increased, notably through a process of differentiation and
specialization rather than proliferation of cells
(Genovese et al., 2000
).
Following transfer to concentrated seawater, the sub-apical space expanded and
septate junction depth was even less than that in normal seawater
(Luquet et al., 2002a
),
indicating further specialization for hypo-osmoregulation.
Superimposed upon these long-term changes in morphology are likely to be
short-term changes in the function and/or expression of transport systems
within the gill epithelium, controlled by hormonal processes or by direct
response to osmotic changes. Indeed, we have shown that alteration of the
osmotic concentration of perfusion media leads rapidly to adaptive changes in
the transport capacity of isolated posterior gills of C. granulatus
(Tresguerres et al., 2003).
Furthermore, dopamine administration to isolated gills results in rapid
changes in transport as well, probably acting through two different receptors,
one activating and one inhibitory
(Halperin et al., 2004
).
Possible targets of these regulatory processes have been tentatively
identified through an analysis of the transport properties of isolated
split-gill lamellae mounted in Ussing-type chambers
(Onken et al., 2003). Ion
substitution and inhibitor application strongly supported a significant role
of basolateral Na+/K+-ATPase in hyper-osmoregulatory ion
uptake in C. granulatus, confirming related studies in this
(Genovese et al., 2004
) and
several other crab species (Burnett and
Towle, 1990
; Lucu and Towle,
2003
; Towle and Kays,
1986
). In addition, experiments with split gill lamellae pointed
toward possible roles in ion uptake for an apical Na+/H+
exchanger, Na+/K+/2Cl- cotransporter and
Cl-/HCO3- exchanger, as well as intracellular
carbonic anhydrase (Onken et al.,
2003
), in line with other models of NaCl absorption across
crustacean gill (Lucu, 1993
;
Onken and Riestenpatt, 1998
;
Towle and Weihrauch, 2001
). In
addition, recent experiments support a role for the V-type
H+-ATPase in energizing Cl- uptake across C.
granulatus gills (G. Genovese and C. M. Luquet, unpublished).
However, very little is known about possible mechanisms of NaCl excretion
resulting in hypo-osmoregulation. A study of the hyper-/hypo-osmoregulating
mangrove crab Ucides cordatus identified differences between
individual gills in their capacity for ion absorption versus ion
excretion (Martinez et al.,
1998), suggesting that the molecular machinery implementing
absorption and excretion is functionally unique. By contrast, Luquet et al.
(2002b
) have recorded similar
ouabain-sensitive potential differences in the three posterior gills of C.
granulatus. Although models for ion excretion across fish gills are quite
well accepted (Evans, 2002
;
Perry, 1997
), no similar
conceptual basis exists for hypo-osmoregulating crustaceans.
To further elucidate the molecular physiology of bidirectional ion
transport across gills of a strongly euryhaline crab, we sought to identify
and characterize the expression of candidate transporter genes in
30-acclimated C. granulatus transferred for varying lengths
of time to dilute (2
) or concentrated (45
) seawater,
hypothesizing that transporters playing an essential role in hyper- or
hypo-osmoregulation might be upregulated via transcriptional
induction. Several candidate transporters in osmoregulatory tissues of
euryhaline crustaceans have been identified and characterized at the molecular
level, including the Na+/H+ exchanger
(Towle et al., 1997a
),
Na+/K+-ATPase
-subunit
(Towle et al., 2001
), V-type
H+-ATPase B-subunit (Weihrauch
et al., 2001
) and Na+/K+/2Cl-
cotransporter (Towle et al.,
1997b
). In the present study, we used quantitative polymerase
chain reaction (QPCR) techniques to analyze mRNA transcript abundance for the
latter three transporters in gills of C. granulatus challenged by
salinity change, to determine if transcriptional expression of
transporter-encoding genes is altered in response to salinity stress. A
predicted housekeeping mRNA, that coding for arginine kinase
(Kotlyar et al., 2000
), was
used as a reference transcript.
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Materials and methods |
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For salinity acclimation experiments, animals were transferred from
30 seawater, in which the hemolymph is essentially isosmotic to the
medium (Luquet et al., 1992
),
to salinities of either 2
or 45
. Crabs were rapidly sacrificed
at timed intervals following the transfer. After removing the dorsal carapace,
gill pairs 3-8 were excised at their base and placed into RNase-free vials
containing a large excess of RNAlater (Ambion, Austin, TX, USA) to
inactivate endogenous RNases in the gill tissue. Tissues in RNAlater
were stored at -20°C until air transport to Mount Desert Island Biological
Laboratory, where they were returned to -20°C.
Total RNA was prepared by pooling the gills of each pair from four animals
(Chomczynski and Sacchi, 1987)
using RNase-free disposable labware with the RNAgents Total RNA kit (Promega,
Madison, WI, USA). RNA quality and quantity were determined by microfluidic
electrophoresis with the RNA 6000 Nano Assay system and 2100 Bioanalyzer
(Agilent, Waldbronn, Germany). Ribosomal RNA produced three sharp peaks (one
18S rRNA and two 28S fragments) characteristic of crustacean and other
arthropod species (Skinner,
1968
).
cDNA was reverse transcribed from mRNA in 2.0 µg of total RNA using
SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and
oligo-dT as primer. Degenerate oligonucleotide primers for the polymerase
chain reaction were based on conserved regions identified by multiple
alignments of target amino acid sequences from other species and were
synthesized by Integrated DNA Technologies (Coralville, IA, USA)
(Table 1). Target cDNAs were
those encoding arginine kinase, a putative housekeeping gene
(Kotlyar et al., 2000), plus
candidate ion transporters V-type H+-ATPase (B-subunit),
Na+/K+/2Cl- cotransporter and
Na+/K+-ATPase (
-subunit). Conventional PCR was
performed at an annealing temperature of 45°C using RedTaq polymerase
(Sigma, St Louis, MO, USA), and amplification products were isolated
electrophoretically on 0.8% agarose gels in TBE buffer.
|
Following gel extraction (Qiagen, Valencia, CA, USA), amplification
products were sequenced on an ABI 3100 automated sequencer (Applied
Biosystems, Foster City, CA, USA) at the Marine DNA Sequencing and Analysis
Center at the Mount Desert Island Biological Laboratory. Raw sequence traces
were analyzed and trimmed using Chromas software
(http://www.technelysium.com.au/chromas.html)
and were submitted to BLASTX analysis for tentative functional identification
(Altschul et al., 1997).
Species-specific primers based on the resulting sequences were designed with
Primer Premier software (Premier Biosoft, Palo Alto, CA, USA)
(Table 1). Open reading frames
were identified, and nucleotide sequences were translated to their most likely
amino acid sequences using DNASIS software (Molecular Biology Insights,
Cascade, CO, USA). Multiple alignments were generated with Multalin
(Corpet, 1988
) and GeneDoc
software
(http://www.psc.edu/biomed/genedoc).
QPCR was accomplished with species-specific primers
(Table 1) at an annealing
temperature of 55°C on a Stratagene MX4000 real-time sequence detection
instrument, using reagents in the Brilliant SYBR Green QPCR kit (Stratagene,
LaJolla, CA, USA). mRNA expression levels were measured in triplicate samples
of cDNA reverse transcribed from 0.10 µg total RNA, thus normalizing to
total RNA levels in each preparation, an accepted method of normalization for
gene expression studies (Bustin,
2002). Relative mRNA abundance was calculated by comparison to a
dilution series of a selected reference cDNA (usually gill 6 from
30
-acclimated animals). Means of relative expression values for
anterior gills 3, 4 and 5 and posterior gills 6, 7 and 8 were pooled for
calculation of overall anterior and posterior gill means and standard errors.
Differences in relative abundance were compared by two-way analysis of
variance (ANOVA) and post hoc comparisons, taking sampling time and
gill group (anterior vs posterior) as factors.
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Results |
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By contrast, the expression of Na+/K+/2Cl-
cotransporter transcripts increased 10-22-fold in posterior gills by 24 h of
exposure to 2 salinity (P<0.001, N=3), with more
modest increases in anterior gills (P<0.05 between gill groups)
(Fig. 4C). The large standard
error noted at 48 h for posterior gills reflects the disparate responses of
the three gills to salinity reduction at that time. Gill 7 contained very high
levels of cotransporter mRNA at 48 h, while gill 8 showed little change from
the previous time sample. However, by 96 h, differences between posterior
gills were smaller, and a 10-fold increase in expression relative to
30
controls was maintained through the 8-day time sample.
Within 6 h after transfer to low salinity,
Na+/K+-ATPase -subunit transcripts increased
5-fold in the three posterior gills but remained unchanged in anterior
gills. However, by 24 h after the transfer to 2
salinity,
-subunit mRNA levels in all tested gills increased between 20- and
50-fold (P<0.001, N=3), with anterior gills declining by
96 h but posterior gills remaining high (P<0.001 between groups)
(Fig. 4D).
Following transfer of crabs from 30 to 45
seawater, a
condition in which C. granulatus is a strong hypo-osmoregulator,
transporter and housekeeping transcript levels showed little change until 96 h
after the transfer (Fig. 5). At
that time, major increases in mRNA abundance for all four target genes
occurred, primarily in posterior gills 6 and 7. The degree of change in
posterior gills was 2-3-fold for arginine kinase (P<0.0001 for
time; P<0.05 between gill groups), 10-fold for V-type
H+-ATPase B-subunit (P<0.0001 for time; no significant
differences between groups), 60-fold for
Na+/K+/2Cl- cotransporter
(P<0.0001 for time; P<0.005 between groups), and
28-fold for Na+/K+-ATPase
-subunit
(P<0.001 for time; P<0.05 between groups)
(Fig. 5A-D). After 8 days, the
expression of all transcripts was similarly high in the three posterior gills
and significantly higher than in anterior gills for
Na+/K+/2Cl- cotransporter,
Na+/K+-ATPase
-subunit and arginine kinase but
not for V-type H+-ATPase B-subunit.
|
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Discussion |
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The Na+/K+/2Cl- cotransporter occurs in
two major forms in vertebrate epithelial cells, an apical form involved in
NaCl uptake and a basolateral form involved in NaCl excretion
(Mount et al., 1998). Ion
substitution experiments in split gill lamellae of Carcinus maenas
support the existence of an apical
Na+/K+/2Cl- cotransporter in this species
(Riestenpatt et al., 1996
).
Previous molecular cloning experiments showed that a
Na+/K+/2Cl- cotransporter is expressed in
gills of crustacean species, including C. granulatus
(Luquet et al., 2003
;
Towle and Peppin, 2002
), but
the resulting sequence information was insufficient to classify the product as
apical or basolateral. The apparent induction of
Na+/K+/2Cl- cotransporter mRNA accumulation
in gills of C. granulatus transferred from 30
to 2
salinity suggests that an apical form may be induced in 2
, where it
could function in NaCl uptake from the medium. On the other hand, an even
stronger apparent induction in 45
, a condition in which the crab is
excreting NaCl most likely via the gills, suggests recruitment of a
basolateral Na+/K+/2Cl- cotransporter,
functioning to move NaCl from hemolymph into branchial epithelial cells, which
would then excrete Na+ or Cl- into the medium. Further
work is required to distinguish between these possibilities.
The most dramatic change in transcript abundance was observed for the
Na+/K+-ATPase -subunit. Between 6 h and 24 h
following transfer from 30 to 2
salinity, the mRNA abundance for the
-subunit reached maximum levels, an approximately 50-fold increase in
posterior gills, and remained at those levels for at least 4 days. Anterior
gills also showed large increases, as much as 20-fold higher than the
30
controls, beginning at 24 h. By contrast, major changes in
Na+/K+-ATPase
-subunit transcript quantity
following transfer to 45
occurred only after 96 h of exposure, similar
to the time frame observed for the
Na+/K+/2Cl- cotransporter.
The Na+/K+-ATPase of crustacean gills is restricted
to the basolateral membrane (Towle and
Kays, 1986), where it can be inhibited in perfused gills by the
specific inhibitor ouabain (Burnett and
Towle, 1990
). In C. granulatus, we have shown that
ouabain produces a large decrease in the transepithelial potential in
symmetrically perfused posterior gills, indicating that the
Na+/K+-ATPase is essential in energizing transbranchial
ion transport (Luquet et al.,
2002b
). A recent study from our laboratory showed that posterior
gills 6, 7 and 8 of C. granulatus contain about 82% of the total gill
Na+/K+-ATPase activity
(Genovese et al., 2004
), a
finding that is reflected in the current study showing a substantially higher
content of Na+/K+-ATPase-encoding mRNA in posterior
gills compared with anterior gills.
However, measurements of Na+/K+-ATPase enzymatic
activity have not shown large differences in C. granulatus gills in
relation to acclimation salinity (Genovese
et al., 2004). Homogenates of posterior gills from animals
acclimated to 30 or 45
seawater exhibit approximately the same
specific Na+/K+-ATPase activity, and animals acclimated
to 10
show only a modest increase
(Genovese et al., 2004
). The
discrepancy between activity measurements and
-subunit mRNA abundance
as detected by quantitative PCR requires explanation. It is known that
catalytic activity of the Na+/K+-ATPase in other species
may be modified by protein kinase A and protein kinase C, as well as by
interaction with a regulatory
-subunit
(Therien and Blostein, 2000
).
In addition, access to the active site and/or the ouabain binding site in
vesicular forms of the enzyme may not occur fully in assays employing
homogenates without detergent treatment
(Lucu and Flik, 1999
).
Hydrolytic activity measured in vitro may therefore not reflect
accurately the functional rate of transport mediated by the
Na+/K+-ATPase. Furthermore, transepithelial potential
differences and 22Na transport in posterior gills of crabs
acclimated to 45
seawater are strongly inhibited by ouabain,
suggesting that ion excretion through C. granulatus gills is indeed
energized by Na+/K+-ATPase
(Luquet et al., 2002b
), a
function that would be enhanced by the accumulation and translation of
-subunit mRNA. The large apparent increases in
Na+/K+-ATPase
-subunit mRNA observed in this
study may reflect a rapid turnover of Na+/K+-ATPase
protein in gill plasma membranes of C. granulatus, requiring a high
level of mRNA to support efficient replacement of the protein.
The rapid response of Na+/K+/2Cl-
cotransporter and Na+/K+-ATPase -subunit mRNA
levels to salinity dilution, compared with the relatively delayed response
observed in hypersaline conditions, probably indicates the existence of
distinctive regulatory mechanisms as well as transport geometry. In work in
our laboratory with split gill lamellae from C. granulatus acclimated
to 2
salinity (Onken et al.,
2003
), we have observed an almost complete inhibition of the
short-circuit current both in Na+-free and Cl--free
medium, along with a strong inhibitory effect of apical CsCl (an inhibitor of
K+ channels). We have concluded that coupled electrogenic NaCl
absorption is mediated by an apically located
Na+/K+/2Cl- cotransporter in parallel with
K+ channels and is energized by Na+/K+-ATPase
at the basolateral membrane. The dramatic induction of
Na+/K+/2Cl- cotransporter and
Na+/K+-ATPase gives further support to this hypothetical
mechanism.
In recent unpublished experiments, we have changed the perfusion conditions
to hypo-osmotic and found an Na+-independent transepithelial
potential difference that is inhibited by the V-type H+-ATPase
inhibitor bafilomycin (G. Genovese and C. M. Luquet, unpublished). These
preliminary data support a role for the V-type H+-ATPase in ion
uptake from extremely dilute salinity (2), supported by the modest
increase in V-type H+-ATPase mRNA measured in the present
study.
The induction of the three candidate transporters after acclimation to high
salinity makes us speculate that they are intimately involved in ion excretion
across the gill. The current model of NaCl excretion in marine fishes includes
a basolateral Na+/K+/2Cl- cotransporter that
mediates the flux of Cl- from the blood to the cytosol of the
ion-transporting cell, energized by the Na+/K+-ATPase
(Evans, 2002). In this model,
Cl- leaves the cell through an apical channel, possibly the cystic
fibrosis transmembrane regulator protein
(Singer et al., 1998
). This
Cl- flux generates an outside negative potential difference, which
is believed to drive Na+ through a paracellular route. Our
electrophysiological and ion flux studies performed on isolated perfused gills
of C. granulatus support the involvement of
Na+/K+-ATPase. However, in most experiments we have
recorded an outside positive potential difference, suggesting that
Na+ and not Cl- is actively transported
(Luquet et al., 2002b
). Thus,
further studies using split gill lamellae mounted in an Ussing chamber remain
to be carried out in order to propose an ion excretion model for crab
gill.
Three levels of response to salinity change have been observed in the gills
of euryhaline crabs: rapid, over a period of minutes; moderately rapid, over a
period of hours; and slow, over a period of days. The most rapid responses
probably involve protein phosphorylation and/or recruitment of membrane
proteins from intracellular stores
(Halperin et al., 2004). The
slowest responses appear to include structural remodeling of the gill
epithelium associated with cellular differentiation
(Genovese et al., 2000
;
Luquet et al., 2002a
). It is
clear that the responses noted here are in the order of hours to days,
providing an intermediate time scale of regulation associated with a complex
pattern of specific transcriptional events.
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Acknowledgments |
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Footnotes |
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