Isolation of a novel aquaglyceroporin from a marine teleost (Sparus auratus): function and tissue distribution
1 Centre of Marine Sciences (CCMAR), Universidade do Algarve, Campus de
Gambelas, 8005-139 Faro, Portugal
2 Center of Aquaculture-IRTA, 43540-San Carlos de la Rapita, Tarragona,
Spain
3 Department of Cell Physiology, University Medical Center St Radboud,
6500HB Nijmegen, The Netherlands
* Author for correspondence (e-mail: dpower{at}ualg.pt)
Accepted 5 January 2004
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Summary |
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Key words: aquaporin, GLP, in situ localisation, multiple transcripts, Fugu rubripes, gastrointestinal tract, kidney, teleost fish, sea bream, Sparus auratus
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Introduction |
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Several CHIP homologues have been identified in mammals: AQP0, AQP1 and
AQP2 (Lanahan et al., 1992;
Preston and Agre, 1991
;
Yang et al., 1999
), AQP4, AQP5
and AQP6 (Krane et al., 1999
;
Raina et al., 1995
;
Sobue et al., 1999
;
Turtzo et al., 1997
;
Yasui et al., 1999
) and AQP8
(Ishibashi et al., 1997a
;
Koyama et al., 1998
). Most of
them have been shown to stimulate osmotic water permeability of plasma
membrane when expressed in Xenopus oocytes. Four aquaporin homologues
belonging to the GLP group have been identified in mammals: AQP3
(Echevarria et al., 1994
;
Ishibashi et al., 1994
;
Ma et al., 1994
), AQP7
(Ishibashi et al., 1997b
),
AQP9 (Kuriyama et al., 1997
;
Tsukaguchi et al., 1998
) and
AQP10 (Hatakeyama et al.,
2001
; Ishibashi et al.,
2002
). Members of the GLP group have a widespread distribution in
tissues associated with water transport. For example, AQP3 is abundantly
expressed in the principal cells of the mammalian kidney
(Ecelbarger et al., 1995
;
Echevarria et al., 1994
), in
the large airways, urinary bladder, conjunctiva, epidermis
(King et al., 1997
;
Nielsen et al., 1997
) and
gastrointestinal tract (Koyama et al.,
1999
; Ramirez-Lorca et al.,
1999
). AQP7 is abundantly expressed in the rat seminiferous
tubules and plasma membrane of late spermatids and is also present in rat
heart, skeletal muscle and kidney
(Ishibashi et al., 1997a
).
AQP9 is expressed at high levels in liver and testis and at lower levels in
brain (Tsukaguchi et al.,
1998
) and AQP10 is expressed in the small intestine
(Ishibashi et al., 2002
).
Relatively few reports about AQP proteins in non-mammalian vertebrates
exist. In amphibians AQP1AQP4
(Abrami et al., 1994;
Ma et al., 1996
;
Schreiber et al., 2000
) have
been identified. In fish, a CHIP protein (AQP0) has been identified in
Fundulus heteroclitus (Virkki et
al., 2001
) and recently an AQP3 homologue was reported in eel
Anguilla anguilla (Cutler and
Cramb, 2002
). Fish are a useful group in which to study aquaporin
evolution as they inhabit diverse aquatic environments and face a constant
osmotic and ionic challenge, which varies according to the waters they inhabit
(freshwater <0.1 mOsmol kg1; seawater approximately 1000
mOsmol kg1). The importance of water and ion transport by
renal and extrarenal epithelial tissues for maintenance of osmotic homeostasis
in fish is well documented (Karnaky,
1998
). However, the importance of the aquaporins in water
movements and associated osmotic homeostasis in fish has received surprisingly
little attention. The present paper reports the cloning and functional
characterisation of an aquaglyceroporin cDNA from a marine teleost, the
gilthead sea bream (Sparus auratus L.). In addition, northern
blotting and in situ hybridisation were used to characterise its
tissue and cellular distribution.
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Materials and methods |
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Construction and screening of sea bream kidney cDNA library
Total RNA was extracted from the collected tissue using `TRI reagent'
(Sigma-Aldrich, St Louis, MO, USA). The poly(A)+ RNA fraction was
obtained from total RNA by chromatography on columns of oligo-dT cellulose
(Amersham Biosciences, Lisbon, Portugal).
A cDNA library was constructed from 5 µg of gilthead sea bream kidney
poly(A)+ RNA using the UNI-ZAP XR cDNA cloning kit according to the
manufacturer's instructions (Stratagene, La Jolla, CA, USA). The
double-stranded cDNA was ligated into the corresponding vector and packaged
into Gigapack Gold III packaging extracts (Stratagene). The sea bream kidney
library was screened with a 700 bp [-32P]dCTP-labelled sea
breamAQP cDNA probe (Rediprime, random labelling kit, Amersham Biosciences)
generated by RT-PCR. Filters were hybridised overnight at 65°C in
ChurchGilbert buffer (1 mmol l1 EDTA, 0.25 mol
l1 NaHPO4, 7% SDS) and washed 2x 10 min at
65°C in 0.1x SSC/0.1%SDS (1/200 dilution of a solution of 3 mol
l1 NaCl, 0.3 mol l1 sodium citrate, pH 7.0
20x SSC). One positive plaque was isolated out of 400 000
recombinants, automatically excised into pBluescript (Stratagene) and
sequenced to give threefold coverage. The sequence has been deposited in
GeneBank (accession number AY363261).
Osmotic water permeability, glycerol permeability and urea uptake assays in Xenopus oocytes
Capped RNA (cRNA) transcripts corresponding to S. auratus AQP were
synthesized in vitro with T3 RNA polymerase (Promega, Madison, WI,
USA) from XhoI-digested pBluescript vector containing the sbAQP cDNA.
The isolation and microinjection of oocytes have been described previously
(Deen et al., 1994). In brief,
pieces of the ovary from Xenopus laevis were treated with collagenase
A (2 mg ml1; Roche, Holland) in modified Barth's culture
medium (MBS), 0.33 mmol l1 Ca(NO3)2,
0.4 mmol l1 CaCl2, 88 mmol l1
NaCl, 1 mmol l1 KCl, 2.4 mmol l1
NaHCO3, 10 mmol l1 Hepes, pH 7.5, 0.82 mmol
l1 MgSO4, for 2 h at room temperature. After
repeated washing with fresh MBS, ovarian follicles at stages VVI were
selected and equilibrated in MBS at 18°C for 24 h. The resulting intact
follicles and partially denuded oocytes were then injected with either 50 nl
of distilled water (negative control), 50 nl of distilled water containing 10
ng cRNA of sbAQP, or with 1 ng cRNA of human AQP1 as a positive control (data
not shown; Denker et al.,
1988
), and incubated in MBS at 18°C for another 24 h.
Subsequently oocytes were completely defolliculated with watchmaker forceps,
equilibrated again for 24 h at 18°C, and then used in water and solute
transport assays.
The osmotic water permeability (Pf) was measured from
the time course of osmotic oocyte swelling in a standard assay. Oocytes were
transferred from 200 mOsm MBS to 20 mOsm MBS medium at room temperature, and
the swelling of the oocytes was followed by video microscopy using serial
images at 2 s intervals during the first 20 s period. The
Pf values were calculated taking into account the
time-course changes in relative oocyte volume
[d(V/Vo)/dt], the oocyte surface area
(S), and the molar volume of water (Vw=18
cm3 ml1), using the formula
Vo[d(V/Vo)/dt]/[SxVw(OsminOsmout)].
To determine glycerol permeability, the oocytes were transferred to an
isotonic solution containing 160 mmol l1 glycerol and
complemented with 40 mOsm MBS to adjust the solution to 200 mOsm. The apparent
glycerol permeability coefficient
() was calculated from oocyte
swelling using the equation
[d(V/Vo)dt]/(S/Vo)
(Verkman and Ives, 1986
). A
slight shrinkage of control eggs was observed as a consequence of a slight
deviation from isotonicity of the incubation medium.
To examine the effect of mercury on the Pf, the oocytes were incubated in MBS containing 1 mmol l1 HgCl2 for 15 min before the swelling assay, which was also performed in the presence of HgCl2. To determine if the mercurial effect was reversible, the same oocytes were rinsed 3 times in MBS, incubated with 5 mmol l1 mercaptoethanol for 15 min, and subjected to the swelling assays 2 h later.
Urea transport activity mediated by sbAQP was measured by uptake of [14C]urea (Amersham Biosciences). Injected oocytes were transferred to 1 mmol l1 urea containing 2 µCi ml1 (74 kBq ml1) in MBS for 20 min, rapidly rinsed 3 times with ice-cold MBS, and lysed in 10% SDS at room temperature followed by liquid scintillation counting.
Northern blot analysis
Between 2 and 5 µg of mRNA from sea bream liver, kidney,
gastro-intestinal tract (duodenum, midgut, hind-gut and rectum), skin, gill
and skeletal muscle were fractionated on a 5.5% formaldehyde/1.5% agarose gel
in MOPS, transferred to a nylon filter (Hybond-N, Amersham) with 10x SSC
and cross-linked at 80°C for 2 h. Prior to hybridisation, the filter was
washed at 60°C for 20 min in 1x SSC, 0.1%SDS and pre-hybridized in
ChurchGilbert buffer for 2 h at 58°C. Hybridisation was allowed to
proceed overnight at 58°C in fresh pre-hybridization solution containing
the full-length sea bream AQP as the probe, isolated from the kidney library
labelled with [-32P]dCTP (Rediprime, random labelling kit,
Amersham Biosciences). Stringency washes were carried out for 2x 15 min
at 60°C in 1x SSC, 0.1% SDS and then at 65°C for 10 min in
0.1x SSC, 0.1% SDS. The blot was exposed to a Phosphoimager (Biorad,
Hercules, CA, USA) for 7 days. In order to evaluate the relative amounts of
mRNA loaded onto the filter for each tissue, the blot was also hybridized with
a sea bream ß-actin probe (Santos et
al., 1997
) following the same protocol as before but reducing
exposure time to 2 h. Exposure data was analyzed using the Multi-Analyst/PC
software package (Biorad, Lisbon, Portugal).
In situ hybridisation
The distribution of sbAQP mRNA along the gastrointestinal tract, in the
gill and kidneys was further investigated by in situ hybridisation.
The full-length sbAQP cDNA cloned in pBluescript was digested with
BamHI (Promega, Madison, WI, USA) at 37°C for 1.5 h in order to
linearise the DNA. The linearised vector was purified and in vitro
transcription was carried out using 20 units of T7 RNA polymerase in
transcription buffer (Amersham Biosciences) with 2 µl of digoxigenin-RNA
labeling mix (Roche, Lisbon, Portugal), for 2 h at 37°C. The riboprobe
synthesis was stopped with 2 µl of 0.2 mol l1 EDTA,
precipitated with sterile sodium acetate (0.1 volume, 3 mol
l1; pH 5.2) and 75% ethanol (2.5 volumes) and resuspended in
50 µl of water. Riboprobe purity and concentration were determined by
electrophoresis in a 1% agarose gel containing ethidium bromide.
Tissue sections were dewaxed, rehydrated and then pre-hybridised at 55°C for 4 h in hybridisation solution (50% formamide, 4x SSC, 1 mg ml1 torula RNA, 0.1 mg ml1 heparin, 1x Denhart's, 0.1% Tween 20, 0.04% CHAPS). Tissues were then hybridised overnight in a humidified box at 55°C in 40 µl per section of hybridisation solution containing 15 µl ml1 of riboprobe. Controls were pretreated with RNase prior to hybridization with riboprobe, or the riboprobe was excluded from the hybridization. High stringency washes were carried out for 3x 5 min at 55°C with 2x SSC. Tissue sections were then washed 2x 5 min with 2x SSC:0.12% CHAPS at 22°C, followed by a wash for 5 min in 2x SSC:PTw (1:1, v/v) and finally 5 min in PTw. Detection of hybridised probe was carried out using anti-digoxigenin-alkaline phosphatase (AP) Fab fragments (1/100) (Roche). The chromagens for colour detection were NBT (4-nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro 3-indolylphosphate) and colour development was carried out over 217 h at30°C. Stained sections were rinsed in PBS, fixed for 15 min in 4% paraformaldehyde at room temperature, rinsed in PBS and mounted in glycerol gelatine. Sections were analysed using a microscope (Olympus BH2) coupled to a video attachment (Sony 3CCD DXP-930P) linked to a computer for digital image analysis.
Phylogenetic and protein structure analysis
A frequent problem in phylogenetic analysis is the limited representation
of fish sequences. For a more robust phylogenetic analysis, the genome of the
model organism Fugu rubripes
(http://fugu.hgmp.mrc.ac.uk)
was searched for GLP members using the sbAQP. Fugu scaffolds
containing GLP protein amino acid signatures were then analysed using NIX, a
Web tool to view the results of a suite of DNA analysis programs
(http://menu.hgmp.mrc.ac.uk).
The predicted coding regions were extracted and further confirmed using BLAST
(Altschul et al., 1990) against
SWISSPROT and non-redundant TREMBL. The human genome was also analysed for the
presence of GLP homologues. The deduced sbAQP amino acid sequence was aligned
with other available aquaporin amino acid sequences in public databases (see
Table 1) using CLUSTAL X
Version 1.64b (Thompson et al.,
1997
) and the percent identity between different AQP forms was
determined (Table 2).
Phylogenetic trees were generated in PAUP* Version 4.0b
(Swofford, 1998
) to identify
maximum parsimony trees for different hypothesis of relationships using
Allium cepa AQP1 (AF255795) as an outgroup in this analysis. The
sbAQP deduced protein structure was analysed using HMMTOP V.2.0 software
(Tusnady and Simon, 2001
) and
a hydropathy profile obtained using the Kite and Doolittle method
(Kyte and Doolittle,
1982
).
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Results |
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In common with other aquaporins, six transmembrane domains were predicted in sbAQP from the hydropathy profile and are underlined in the deduced aa sequence (Fig. 1). The MIP family signature, Asn-Pro-Ala (NPA), was present at aa 7678 and at aa 208210 of sbAQP, which are located, respectively, in the second (B) and fifth (E) connecting loop. Both the N terminus and C terminus were predicted to be in the cytosol, as for other AQPs. Amino acid residues 8, 18, 33, 84, 167, 244, 272 and 291 were cysteine. No potential N-linked glycosylation site [NX(S/T)] was found in sbAQP. The deduced molecular mass of the protein is 31.6 kDa and the theoretical pI 6.5.
Functional characterisation of sbAQP
Xenopus laevis oocytes injected with cRNA encoding sbAQP had a
sevenfold larger Pf than that of oocytes injected with
water alone (control, Fig. 2A).
Moreover, oocytes expressing sbAQP showed increased Pgly
and Purea (measured, respectively, using volumetric and
radiotracer methods) relative to the values found with water-injected oocytes
(Fig. 2B,C). Comparison of the
relative permeability of sbAQP to water, glycerol and urea revealed that its
functional characteristics are similar to mammalian AQP3 and AQP10: it is most
permeable to water and its permeability to glycerol and urea is several-fold
lower (Echevarria et al.,
1996). The water channel function of sbAQP was sensitive to
mercuric chloride (HgCl2). In the presence of 1 mmol
l1 HgCl2 there was a
70% decrease in osmotic
water permeability (Pf). However, it was not possible to
reverse this effect by mercaptoethanol treatment. This is in contrast to the
observations of human AQP3 and AQP10, where at 1 mmol l1
HgCl2 there is significant inhibition of water transport, which is
partially reversed in the presence of mercaptoethanol
(Ishibashi et al., 2002
;
Kuwahara et al., 1997b
).
|
SbAQP expression and tissue distribution
Two transcripts of 2 kb and 1.6 kb were identified by northern blotting in
the gastrointestinal tract (mid-gut, hind-gut and rectum) and in all these
tissues the relative abundance of both transcripts was similar
(Fig. 3A). In kidney only the
large transcript of 2 kb and in gill only the 1.6 kb transcript were detected.
No signal was detected in duodenum or in any of the other tissue analysed.
Comparison of the expression of sbAQP with that of ß-actin
(Fig. 3B) indicated that
hind-gut and kidney are the tissues with the highest expression of sbAQP
followed by the rectum, mid-gut and gill.
|
In situ hybridisation was carried out to characterise the cellular expression of sbAQP in the gastrointestinal tract, gills and kidney (Fig. 4). An intense sbAQP signal was found in the epithelial cell lining some of the kidney tubules (Fig. 4A,C). In gill sbAQP expression was most intense in the primary filaments, where the chloride cells are normally found, and expression of sbAQP was also observed at the proximal ends of some of the secondary lamellae (Fig. 4D,F). The expression of sbAQP in the hind-gut was principally in cells scattered in the lamina propria (Fig. 4G), although the identity of these cells remains to be established. Further expression of sbAQP was also found in cells localised at the interface of the circular and longitudinal muscle layer in the hind-gut (Fig. 4I). sbAQP was not found to be expressed in either the columnar enterocytes of the villi or in the numerous goblet cells of the hind-gut. A similar pattern of sbAQP distribution was found in the mid-gut, although the signal was far weaker. Despite being unable to detect sbAQP transcripts in the duodenum by northern blot a very weak signal was found in cells of the mucosa by in situ hybridisation (data not shown). No sbAQP signal was detected in the liver (Fig. 4J) and the controls utilised to verify probe specificity were also negative (Fig. 4B,E,H).
|
Phylogenetic analysis
Analysis of the Fugu genome led to the identification in
silico of six scaffolds containing putative GLP genes. The extracted
Fugu genes were introduced in a multisequence alignment
(Fig. 5) used in the
phylogenetic analysis. Two Fugu genes (from scaffolds M000233 and
M004004) were excluded because they were extremely truncated. The first
phylogenetic analysis done showed two principal clades that corresponded,
respectively, to GLP and CHIP proteins. To better exemplify the relationship
between the GLP family members, phylogenetic analysis was performed with only
members of this group and Allium cepa AQP1 was the outgroup
(Fig. 6). The GLP clade was
composed of separate groups that corresponded to AQP3, AQP7, AQP9 and AQP10.
The Fugu gene in scaffold M001883 grouped with AQP3 and was most
similar to the available fish sequences. The genes in scaffolds M000386 and
M001042 grouped, respectively, with mammalian AQP7 and AQP10. AQP9 was the
only GLP in which there was no match with a predicted Fugu GLP gene.
The sbAQP formed an independent group clustering with the Fugu gene
from scaffold M000648. Extensive searches of the human genome failed to
identify a sbAQP paralogue.
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Discussion |
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Sequence similarity and phylogenetic analysis incorporating extracted Fugu genes, and the identification of a Fugu gene that clustered in an independent group with sbAQP, strongly suggest that sbAQP is a novel GLP member. Extensive searches of the human genome failed to identify a paralogue, suggesting that the gene found in sea bream and Fugu may have evolved only in the fishes after divergence from the tetrapod lineage.
Characterisation of sbAQP function using the Xenopus oocyte water
and solute transport assay indicates that it is a functional fish
aquaglyceroporin and transports water, glycerol and urea. Moreover, the
osmotic water permeability is mercury (HgCl2) sensitive. However,
treatment with mercaptoethanol fails to reverse the latter effect and it is
unclear if this is an artefact caused by non-specific effects, such as the
toxicity of the high-dose of HgCl2 (1 mmol l1),
on Xenopus oocyte viability or reflects a characteristic of the sbAQP
protein (Kuwahara et al.,
1997b). Mercurial reagents are thought to block the aqueous pore
of AQPs by binding specifically to cysteine residues, and Cys-11 in mammalian
AQP3 and Cys-212 in mammalian AQP2 have been shown to confer this
characteristic (Kuwahara et al.,
1997a
). The absence of Cys-11 but presence of Cys-8 in sbAQP may
indicate that the latter is responsible for the observed mercury sensitivity,
although candidate cysteine residues are present nearby at positions 18, 22
and 33.
Analysis of sbAQP tissue distribution in adult sea bream, demonstrated that
it is widely distributed in water-transporting epithelia. The gastrointestinal
tract, but more specifically the hind-gut and the kidney, express the highest
levels of sbAQP mRNA. The origin of the two transcripts identified in the sea
bream gastrointestinal tract has not been determined, although the relatively
limited conservation of sbAQP with other members of the GLP group isolated so
far in vertebrates (Table 2),
and the high stringency conditions utilised for the northern blot, appear to
rule out probe cross-hybridisation with these forms. However, identification
in silico in the Fugu genome of at least nine AQP raises the
possibility that the probe used in the present study could have
cross-hybridised with an as-yet-unknown isoform and further work will be
required to resolve this question. In human, two forms of AQP10 have been
identified, a 2 kb transcript and a splice variant of 2.3 kb that lacks
the sixth transmembrane domain (Hatakeyama
et al., 2001
; Ishibashi et
al., 2002
). Multiple transcripts of another GLP family member,
AQP7, have also been reported in rat with two transcripts found in kidney,
heart and skeletal muscle (Ishibashi et
al., 1997a
). It will be of interest to determine the nature of the
two transcripts of AQP identified in sea bream and to characterise their
function in vivo. For example, in man the 2.3 kb splice variant of
AQP10 lacking the sixth transmembrane domain is permeable to water but not to
glycerol or urea, unlike the full-length 2 kb transcript.
In man the duodenum and jejunum are the major sites of expression of AQP10,
and these are also the sites for entrance and secretion of large volumes of
water (Hatakeyama et al.,
2001). A similar observation is made with AQP3, which is expressed
in the basolateral membrane in the collecting duct of kidney
(Ecelbarger et al., 1995
;
Echevarria et al., 1994
;
Ma et al., 2000
) and in the
rectum and hind-gut (Ramirez-Lorca et al.,
1999
). The general presence of AQPs in membranes important for
water movement has led to the suggestion that transcellular as well as
paracellular pathways of water movement exist in this tissue in man. The
behaviour of aquaporins reported in mammals and their response to movements of
water driven by osmotic gradients, fits in well with experimental observations
of water movement in the fish intestine, where it has been linked to ion
movements similar to those reported in ouabain-sensitive water absorption in
Atlantic salmon (Usher et al.,
1991
) and Fundulus heteroclitus (Marshall et al.,
2002a
,b
).
In the latter situation it has been demonstrated that the intestine not only
absorbs water but also secretes fluid by mechanisms linked, at least in part,
to cystic fibrosis transmembrane conductance regulator-like (CFTR-like) ion
channel. More studies will be required to determine if sbAQP is directly
involved in water absorption in the intestine in order to establish whether it
provides a mechanism by which drinking and water absorption can be
independently regulated in the fish intestine
(Fuentes and Eddy, 1997
).
In teleost fish the only members of the MIP family identified are an AQP0
homologue in the killifish Fundulus heteroclitus (Accession number,
AF191906), AQP3 homologues in the European eel Anguilla anguilla
(Accession number, AJ319533) and zebra fish Danio rerio (Accession
number, BC044188), and an AQP in Tribolodon hakonenis (Accession
number, AB055465) (Cutler and Cramb,
2002; Virkki et al.,
2001
). In killifish AQP0 is restricted to the lens and is present
as two transcripts of 2.8 and 1.8 kb, which are suggested to correspond to
alternative splice variants. AQP3 in the European eel has a wider tissue
distribution, being present in low abundance in the eye, oesophagus and
intestine as a single transcript of 2.4 kb and in high abundance in the gills,
where an additional transcript of 7 kb is also identified. Moreover, in the
eel the level of expression of AQP3 appears to be regulated by the composition
of the bathing medium, so that the expression of AQP3 in the gills of
freshwater-adapted eel is several-fold higher than in those adapted to
saltwater. Comparison of the eel AQP3 and sbAQP tissue distribution reveals an
overlap, although the relative abundance of transcripts seems to differ. The
gill in freshwater fish plays an important role in total body water influx and
is suggested to account for up to 90% of water uptake
(Haywood et al., 1977
).
However, this is not true in marine fish where the presence of leaky epithelia
ensures high water loss through the gills, in particular the chloride cell
complex. The distribution of sbAQP mRNA in the gills of the sea bream
coincided with that described by immunohistochemistry for AQP3 in
seawater-adapted European eel (Lignot et
al., 2002
). The mRNA of AQP in seabream and protein of AQP3 in
European eel is principally localised in the chloride cells within the primary
filaments of the gills and at the proximal ends of the secondary lamellae. The
physiological role of sbAQP in the gill remains to be established, whether it
is directly involved in water movement and/or also associated with elimination
of nitrogenous waste products (Isaia,
1984
).
The tissue distribution of sbAQP coincides with that of the mammalian GLP
proteins, AQP3, AQP7 and AQP10. Surprisingly the expression of eel AQP3 in the
kidney is very low and does not coincide with the abundant expression in the
basolateral membrane of the collecting duct
(Ecelbarger et al., 1995;
Echevarria et al., 1994
;
Ma et al., 2000
) in mammals.
In seawater fish such as the sea bream, the kidney is unable to produce highly
concentrated urine and instead produces low quantities of urine containing
abundant quantities of divalent ions such as Mg2+, Ca2+
or even SO 2 4. The relatively high expression of sbAQP in
the kidney may be related to functional adaptations in the kidney of seawater
fish, where water movements are thought to be linked to monovalent ions such
as Na+, Cl resorption and secretion in the
tubules (Beyenbach and Liu,
1996
).
In conclusion, a sbAQP cDNA was isolated from a kidney cDNA library and its primary structure was found to be homologous to other GLP proteins, with the same functional characteristics. Phylogenetic analysis of sbAQP, including putative GLP genes from Fugu, demonstrated that it did not group with any of the previously isolated GLP and instead formed a separate group, suggesting that it may be a novel GLP member.
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Acknowledgments |
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References |
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