Aquaporin-3 expressed in the basolateral membrane of gill chloride cells in Mozambique tilapia Oreochromis mossambicus adapted to freshwater and seawater
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
* Author for correspondence (e-mail: watanabe{at}marine.fs.a.u-tokyo.ac.jp)
Accepted 10 May 2005
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Summary |
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Key words: aquaporin, gill, osmoregulation, tilapia, Oreochromis mossambicus
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Introduction |
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Aquaporins (AQPs) are a family of integral membrane proteins that function
as water-selective channels, and are importantly involved in fluid
transporting mechanisms in various organs
(Agre et al., 2002;
King et al., 2004
). More than
ten AQPs have been identified in mammals; deduced amino acid sequences and the
predicted six-transmembrane-spanning topology are highly conserved between
species and between family members. Among AQP isoforms, AQPs 0, 1, 2, 4 and 5
are considered to be water-selective (Agre
et al., 1993
; Hasegawa et al.,
1994
; Raina et al.,
1995
; Yang and Verkman,
1997
), whereas AQPs 3, 7, 9 and 10 are also permeable to glycerol
and other small nonelectrolyte solutes, often referred to as
`aquaglyceroporins' (Ishibashi et al.,
1994
,
1997
,
1998
,
2002
;
Echevarria et al., 1996
). AQP6
has been proposed to function in kidney endosomes as a pH-sensitive anion
channel (Yasui et al.,
1999a
,b
).
In teleost fishes, several AQP homologues have been cloned to date, including
AQP0 from the lens of killifish Fundulus heteroclitus
(Virkki et al., 2001
), AQP1
from the intestine of Japanese eel Anguilla japonica
(Aoki et al., 2003
), sbAQP from
the kidney of sea bream, Sparus auratus
(Santos et al., 2004
), and
AQP3 from the gills of European eel Anguilla anguilla
(Cutler and Cramb, 2002
), and
Japanese dace Tribolodon hakonensis
(Hirata et al., 2003
).
Although AQP3 may be involved in water movements and the related biological
events in gills of teleosts, its physiological significance remains unclear
(Cutler and Cramb, 2002
;
Lignot et al., 2002
;
Hirata et al., 2003
).
In this study, we attempted to search for AQP3 in the gills of Mozambique tilapia by PCR-based cloning in order to gain a better understanding of the mechanism and route of water movements. Here we report the cloning, tissue distribution, functional expression, and cellular and intracellular localization of tilapia AQP3. Our findings suggested the involvement of AQP3 in the mechanisms of volume regulation and osmoreception in gill chloride cells.
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Materials and methods |
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Molecular cloning of tilapia AQP3 cDNA
Cloning of the partial-length cDNA
Freshwater-adapted tilapia was used for total RNA preparation. After
anesthesia, the gills were dissected out and frozen in liquid nitrogen. Total
RNA was extracted with RNA extraction solution (ISOGEN; Nippon Gene, Toyama,
Japan) from the gills. Total RNA was treated with DNase (Promega, Madison, WI,
USA) at 37°C for 1 h, and 2 µg total RNA was reverse-transcribed using
Ready-To-GoTM T-Primed First-Strand cDNA Synthesis Kit (Amersham
Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer's
instructions.
Degenerate primers used for amplification were designed based on the available information in vertebrate species. Reverse transcription (RT)-PCR was carried out using sense (AQP3-df) and antisense (AQP3-dr) primers (Table 1). PCR was performed in a final volume of 20 µl containing 1 x PCR buffer (TaKaRa, Shiga, Japan), 200 µmol l1 of dNTPs (TaKaRa), 0.5 U Taq DNA polymerase (TaKaRa r Taq; TaKaRa), 5 µmol l1 each of the primer pair, and an appropriate amount of the gill cDNA template. The PCR cycle protocol was as follows: 94°C for 3 min, 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with final incubation at 72°C for 7 min. The PCR products were analyzed on a 2% agarose gel and ligated into pBluescript SK () (Stratagene, La Jolla, CA, USA). The plasmid DNA was purified, and both strands of the DNA were sequenced using a DNA sequencer ABI PRISM 310 (Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed with Sequencher software version 3.1.1 (Hitachi, Tokyo, Japan). All primers using for PCR-based cloning are listed in Table 1.
|
3' Rapid amplification of cDNA end (RACE)
After determination of the partial cDNA sequence, 3' rapid
amplification of cDNA end (RACE) was carried out to extend sequence
information at the 3' end. The 3' end of tilapia AQP3 cDNA was
amplified with a gene-specific primer, 3' AQPf-f, and an adaptor primer,
RTG (Table 1), in a reaction
mixture (20 µl) containing 1 x PCR buffer, 200 µmmol
l1 dNTPs, 0.5 U Taq DNA polymerase (TaKaRa r
Taq; TaKaRa), 0.25 µmol l1 primers, and 2 µl of
tenfold diluted cDNA template. The PCR cycle protocol was as follows: 94°C
for 3 min, 38 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for
90 s, with final incubation at 72°C for 4 min. The clone obtained was
subcloned and sequenced as described above.
5' RACE
Total RNA was isolated as described above from the gills of FW-adapted
tilapia. Total RNA (2 µg) was reverse-transcribed, and 5' RACE-ready
first-strand cDNA was synthesized using SMARTTM RACE cDNA Amplification
Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's
instructions. For 5' RACE, an adaptor primer, NUP, and a gene-specific
primer, 5' AQPf-r, were used (Table
1). The 5' RACE-PCR was carried out with these primers under
the same conditions as 3' RACE-PCR. The clone obtained was subcloned and
sequenced as described above.
Analysis of the amino acid sequence
Kyte-Doolittle hydropathy profile of the deduced amino acid sequence was
analyzed using GENETYX-MAC version 8.0 software (Software Development, Tokyo,
Japan) at a 12-residue window. The amino acid identities between tilapia AQP3
and other AQPs were analyzed by the BLAST search
(Altschul et al., 1997).
Northern blot analysis
To confirm the full-length of tilapia AQP3 mRNA, total RNA was isolated
from the gill of FW-adapted tilapia. Poly(A)+ RNA (3 µg) was
purified using Oligotex-dT30 Super (TaKaRa) and electrophoresed on a 1.2%
formamideagarose gel, and transferred to a Biodyne nylon membrane (Pall
Gelman Sciences, Ann Arbor, MI, USA). The membrane was UV cross-linked (120
000 µJ cm-2) using Spectrolinker XL-1500 (Spectronics
Corporation, Westbury, NY, USA) and dried in a dry oven at 80°C for 15
min. The membrane was prehybridized at 42°C for 3 h in 6 x SSC
containing 50% formamide, 1 x Denhardt's solution, 0.5% SDS and calf
thymus DNA (100 ng ml1). The buffer was then replaced, and
the membrane was hybridized with a [32P]-labeled cDNA probe (see
below) in the same buffer at 42°C for 18 h. After hybridization, the
membrane was washed in 2 x SSC containing 0.1% SDS at room temperature
for 5 min. The membrane was then exposed to an X-ray film RX-U (Fuji Film,
Tokyo, Japan) at 80°C. A cDNA probe for tilapia AQP3 was generated
with the RT-f and 5'AQPf-r (Table
1) from the tilapia AQP3 cDNA clone and radiolabeled with
[-32P]dCTP (Amersham Pharmacia Biotech) by PCR
amplification.
Reverse transcription-PCR (RT-PCR) analysis
The brain, pituitary, kidney, liver, spleen, intestine, eye, gill and skin
were removed from FW- and SW-adapted tilapia, and total RNA was extracted from
these tissues as described above. Total RNA (2 µg) was reverse-transcribed
using SuperscriptTM First-Strand Synthesis System for RT-PCR (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer's instructions. The cDNA was
amplified with a primer pair, RT-f and RT-r
(Table 1). The PCR products
were electrophoresed on a 2% agarose gel and stained with ethidium bromide. As
a negative control, PCR was conducted in the absence of the cDNA template.
Oocyte expression
The posterior fragment of tilapia AQP3 cDNA was amplified using a primer
set, RT-f and AQP3+poly(T) (Table
1), subcloned into pCR 4Blunt-TOPO (Invitrogen), excised from pCR
4Blunt-TOPO using NcoI and PstI, and ligated into pT7Blue-2
(Novagen, Madison, WI, USA). The anterior fragment of tilapia AQP3 cDNA was
amplified using a primer set, AQP3+kozak and 5' AQPf-r
(Table 1), subcloned into
pBluescript SK() (Stratagene), excised from pBluescript SK()
using NcoI, and ligated into pT7Blue-2 containing the posterior
fragment of tilapia AQP3. Capped cRNA was transcribed in vitro using
T7 mMessage mMachine kit (Ambion, Austin, TX, USA) after digestion with
EcoRI to linearize the plasmids.
Xenopus laevis oocytes (stage VVI) were defolliculated with collagenase (Sigma, St Louis, MO, USA) and microinjected with 10 ng of the synthesized cRNA or 50 nl of distilled water as a control, and incubated at 18°C for 48 h in modified Barth's solution (MBS; 88.0 mmol l1 NaCl, 1.0 mmol l1 KCl, 2.4 mmol l1 NaHCO3, 0.3 mmol l1 CaNO3 2, 0.41 mmol l1 CaCl2, 0.82 mmol l1 MgSO4, 1000 IU ml1 penicillin and 100 µg ml1 streptomycin).
Measurement of oocyte water permeability
After 48 h incubation, oocytes were transferred from 200 mOsm
kg1 (Osmin) to 70 mOsm kg1
(Osmout) of MBS diluted with distilled water at 22°C. We
acquired images of the oocyte silhouette every 15 s through a CCD camera
attached to a stereomicroscope (SMZ1500, Nikon, Tokyo, Japan) up to 5 min or
the time of oocyte rupture. The saved images were later analyzed with
Image-Pro® Plus 4.5.1 (Media cybernetics, Silver Spring, MD,
USA). The oocyte volume was calculated from the cross-sectional area of the
oocyte, assuming the oocyte to be a perfect sphere.
The osmotic water permeability (Pf) was calculated using the following
equation (Preston et al.,
1992):
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Effects of mercuric chloride were examined by incubating oocytes in MBS containing 0.3 mmol l1 HgCl2 for 10 min prior to Pf measurements. To examine the recovery of the HgCl2-induced inhibition by a reducing agent, oocytes were incubated for 10 min in MBS containing 5 mmol l1 ß-mercaptoethanol following 10 min incubation in HgCl2. Significant differences in Pf values at P<0.01 were determined by one-way analysis of variance (ANOVA), followed by Fisher's PLSD, using StatView 5.0 software (Hulinks, Tokyo, Japan).
Antibody
A polyclonal antibody was raised in a rabbit against a synthetic peptide
corresponding to part of the C-terminal region of tilapia AQP3 molecules
(amino acid residues 267279 in tilapia AQP3). The antigen conjugated
with keyhole limpet hemocyanin (KLH) was emulsified with complete Freund's
adjuvant, and immunization was performed in a New Zealand white rabbit. The
antiserum was obtained after several booster injections, and the specific
antibody was affinity-purified from the antiserum with the antigen peptide
(QIAGEN, Hilden, Germany).
Western blot analysis
The specificity of the antibody raised against the synthetic peptide was
confirmed by western blot analysis. Prior to western blotting, the sample was
subjected to immunoprecipitation. The gill was isolated from FW-adapted
tilapia, and the gill filaments were scraped in 1 ml of lysis buffer
consisting of IP buffer (pH 7.4; 140 mmol l1 NaCl, 2 mmol
l1 KCl, 10 mmol l1 Hepes, 5 mmol
l1 EDTA), inhibitors (10 mmol l1
benzamidine, 1 µg ml1 Pepstatin A and 2 mmol
l1 phenyl methyl sulfonyl fluoride) and 1% Triton X-100, and
left on ice for 20 min to lyse the cells. The lysate was centrifuged at 5000
g for 5 min at 4°C, and the supernatant was incubated with
2 µl of the tilapia AQP3 antibody at 4°C for 16 h. Slurry (20 µl)
containing 50% protein A sepharose beads (Amersham Pharmacia Biotech) blocked
overnight with 1% bovine serum albumin (BSA) was added to the sample, and the
mixture was incubated for 1 h at 4°C. After washing five times with IP
buffer containing inhibitors and centrifugation at 10000 g for
30 s, 30 µl of hot Laemmli buffer
(Laemmli, 1970) containing 5%
ß-mercaptoethanol was added to beads binding to the antibody, and the
mixture was incubated for 15 min at 65°C. The sample was centrifuged at 10
000 g for 2 min. The supernatant was separated by
SDSpolyacrylamide gel electrophoresis using PAG Mini `DAIICHI' 15/25
(Daiichi pure chemicals, Tokyo, Japan). After electrophoresis, the protein was
transferred from the gel to a polyvinylidene fluoride microporous membrane
(Immobilon-P Transfer Membrane; Millipore, Billerica, MA, USA). After blocking
with a blocking solution (Block Ace; Dainippon Pharmaceutical, Osaka, Japan)
for 1 h at room temperature, the membrane was incubated with anti-tilapia AQP3
diluted 1:100 with 20 mmol l1 Tris-buffered saline
containing 0.05% Tween 20 (TBS-T) for 1.5 h at room temperature. After rinsing
in TBS-T, the membrane was stained by the avidinbiotinperoxidase
complex (ABC) method (Hsu et al.,
1981
), using commercial reagents (Vectastain ABC kit, Vector
Laboratories, Burlingame, CA, USA). Briefly, the membrane was incubated with
biotinylated anti-rabbit IgG for 30 min, and then with ABC for 30 min at room
temperature. The membrane was finally incubated with 0.02%
3,3'-diaminobenzidine tetrahydrochloride (DAB) containing 0.005%
H2O2 for 3 min to visualize the immunoreactive
bands.
Fluorescence microscopy
The gills were dissected out and fixed in 4% paraformaldehyde (PFA) in 0.1
mol l1 phosphate buffer (PB, pH 7.4) for 16 h at 4°C.
After fixation, the gills were dehydrated in ethanol, and embedded in
Paraplast. Sections (4 µm) were cut and mounted on MAS-coated slides
(Matsunami, Osaka, Japan). For the detection of chloride cells in the
sections, we used an antibody specific for
Na+/K+-ATPase. The antiserum (NAK121) was raised in a
rabbit against a synthetic peptide corresponding to part of the highly
conserved region of the -subunit
(Uchida et al., 2000
). The
sections were incubated with anti-tilapia AQP3 diluted 1:200 with PBS
containing 2% normal goat serum (NGS), 0.1% BSA, 0.02% KLH and 0.01% sodium
azide (NB-PBS) overnight at 4°C, and then with goat anti-rabbit IgG
labeled with Alexa Fluor 488 (Molecular probes, Eugene, OR, USA) diluted
1:1000 with NB-PBS for 3 h at room temperature. After rinsing in PBS, the
sections were incubated with Alexa Fluor 546-labeled
anti-Na+,K+-ATPase
(Katoh and Kaneko, 2003
)
diluted 1:2000 with NB-PBS at 4°C for 8 h. The sections were observed
under a fluorescence microscope (Nikon E800) with blue (excitation,
450490 nm; emission, 520560 nm) and green (excitation,
510560 nm; emission, >590 nm) excitation filter blocks for Alexa
Fluor 488 and Alexa Fluor 546, respectively. To confirm the specificity of the
immunoreaction for tilapia AQP3, the sections were incubated with the
preimmune serum instead of anti-tilapia AQP3.
Transmission electron microscopy
The gills were fixed in 2% PFA, 0.2% glutaraldehyde (GA) in 0.1 mol
l1 PB for 8 h at 4°C. After washing in PBS for 1 h, the
gills were immersed in 30% sucrose in PBS for 1 h, and embedded in Tissue-Tek
OCT compound (Sakura Finetek, Tokyo, Japan) at 20°C. Cryosections
(20 µm) were cut on a cryostat (CM 1100, Leica, Wetzlar, Germany) at
20°C, and mounted on MAS-coated slides (Matsunami). The sections
were immunocytochemically stained by the ABC method, using commercial reagents
(Vectastain ABC kit). In brief, the sections were incubated sequentially with:
(1) 0.6% H2O2 for 30 min, (2) 2% NGS for 30 min, (3)
anti-tilapia AQP3 diluted 1:100 at 4°C overnight (for controls, the
pre-immune serum instead of anti-tilapia AQP3), (4) biotinylated anti-rabbit
IgG for 1.5 h, (5) ABC for 1.5 h, and (6) 0.02% DAB containing 0.005%
H2O2 for 7 min. The sections were then post-fixed in 1%
osmium tetroxide in 0.1 mol l1 PB for 10 min. After
dehydration in ethanol, the sections were embedded in Spurr's resin
(Polysciences, Warrington, PA, USA). Ultrathin sections were cut using a
diamond knife and mounted on grids. The specimens were observed using a
transmission electron microscope (JEOL-1010, JEOL, Tokyo, Japan).
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Results |
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Northern blot analysis showed that the hybridized signal was detected as a single band of approximately 1.8 kb in the gill of FW tilapia (Fig. 2). The size of mRNA was in agreement with that of the cDNA for tilapia AQP3 obtained in this study.
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Western blot analysis
In Western blot analysis, the antibody raised against the synthetic peptide
corresponding to part of the C-terminal region of tilapia AQP3 recognized two
protein bands with molecular mass of 26 and 28 kDa
(Fig. 5). The 28 kDa band
corresponding to the mass predicted from the open reading frame of the cloned
cDNA was the major one, while the smaller band of 26 kDa was somewhat
faint.
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Cellular and intracellular localization of tilapia AQP3 in the gills
Na+/K+-ATPase-immunoreactive chloride cells observed
by light-microscopic immunocytochemistry were mostly located in the afferent
vascular half of the gill filaments, whereas they were rarely observed in the
efferent side of the filaments and on the lamellar epithelia
(Fig. 6C,D). Chloride cells
were apparently larger and more intensively stained with
anti-Na+/K+-ATPase in SW-adapted fish than in FW-adapted
fish. The intense immunoreaction for AQP3 was detected in chloride cells in
both FW- and SW-adapted tilapia (Fig.
6A,B). The specific immunoreaction was not observed in the other
part of the gills. The immunoreactions for AQP3 and
Na+/K+-ATPase coincided completely with each other
(Fig. 6E,F). There was no
significant difference in AQP3 immunoreactivity in chloride cells between FW
and SW gills. In the controls, where sections were incubated with the
pre-immune serum, the immunoreaction was not detected in either FW or SW fish
(Fig. 6G,H).
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Electron-microscopic immunocytochemistry further revealed that AQP3 was localized in the basolateral membrane, but not in the apical membrane, of gill chloride cells in FW- and SW-adapted tilapia (Fig. 7A,C). The specificity of the immunoreaction was confirmed by replacement of the specific antibody with the pre-immune serum: the immunoreaction in the basolateral membrane was extinguished, although mitochondria showed some non-specific reaction (Fig. 7B,D).
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Discussion |
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To further evaluate the water-transporting function of tilapia AQP3, the
synthesized cRNA encoding tilapia AQP3 was injected into Xenopus
oocytes to allow its expression, and osmotic swelling was measured in the
oocytes following transfer to the hypoosmotic solution. The AQP3-expressed
oocytes swelled in the hypoosmotic medium, absorbing water according to the
osmotic gradient created between internal and external media, while the
swelling was not evident in the control oocytes. The Pf values calculated from
the time-course changes in oocyte swelling were about 30 times higher in the
cRNA-injected oocytes than in the control. The increase in the Pf value of
cRNA-injected oocytes was about 80% inhibited by pre-treatment with
Hg2+, a potent inhibitor of AQP that combines `mercury-sensitive'
cysteine residues of AQP to block its aqueous pore
(Kuwahara et al., 1997). This
Hg2+-induced inhibition was restored by ß-mercaptoethanol, the
reducing agent that may dissociate Hg2+ from AQP. Thus, the
inhibition by Hg2+ was not due to non-specific toxicity of mercury
compounds, but was a reversible phenomenon. These results indicate that AQP3
expressed in the Xenopus oocytes and incorporated into the vitellin
membrane increased water permeability, providing functional evidence of
tilapia AQP3 as a water channel. Because of high homology with mammalian AQP3,
tilapia AQP3 is also likely to function as an aquaglyceroporin, although we
did not address its glycerol-transporting ability in this study.
In the present study, AQP3 was extensively expressed in various tissues
examined, except for the liver, in tilapia adapted to both FW and SW. It
should be noted that these AQP3-expressing tissues include major
osmoregulatory organs (gills, kidney and intestine), suggestive of its
involvement in osmoregulatory processes. In mammalian species, AQP3 is
ubiquitously expressed in the epithelia of the urinary tract, digestive tract
and respiratory tract and epidermis of the skin. It has been suggested that
mammalian AQP3 is involved in osmoregulation and osmoprotective systems
against dehydration in terrestrial life
(Ma et al., 2000;
Matsuzaki et al., 1999
;
Kreda et al., 2001
;
Hara and Verkman, 2003
).
A specific antibody was raised against tilapia AQP3. In western blot analysis, the antibody recognized one major band of approximately 28 kDa and another faint band of 26 kDa. The 28 kDa band corresponds well to the predicted mass of tilapia AQP3, whereas the minor 26 kDa band may represent a degenerated product of the native AQP3. In spite of the unexpected immunoreactive band of smaller molecular mass, the result suggests high specificity of the antibody and its availability for immunocytochemical detection of AQP3.
The Na+/K+-ATPase is a key enzyme in the
ion-transporting functions of chloride cells in both FW- and SW-acclimated
fish (McCormick, 1995), and
the antiserum specific for this enzyme serves as a specific marker for their
immunocytochemical detection (Ura et al.,
1996
). As Na+/K+-ATPase is located in the
tubular system, which is continuous with the basolateral membrane
(Karnaky et al., 1976
;
Hootman and Philpott, 1979
;
Hirose et al., 2003
), the
widespread distribution of the tubular system in the cytoplasm results in cell
labeling with the nucleus remaining unstained. In the present
light-microscopic immunocytochemistry, the AQP3 immunoreaction coincided
completely with the Na+/K+-ATPase immunoreaction,
suggesting the colocalization of both molecules in the basolateral membrane of
gill chloride cells. The basolateral localization of AQP3 was further
confirmed by the electron-microscopic immunocytochemistry, in which intensive
immunoreactive signals were detected along the membrane of the tubular system.
It is notable that the observed immunoreaction for AQP3 in gill chloride cells
at light- and electron-microscopic levels showed no clear distributional
difference between FW and SW tilapia. This suggests that this molecule is not
directly related to water transport for FW and SW adaptation, but is involved
in more fundamental mechanisms common to chloride cell functions in FW and
SW.
Chloride cells are typically classified into two types on the basis of
their ion-transporting functions; that is, FW-type cells that absorb NaCl in
fish adapted to hypoosmotic environments, and SW-type cells that excrete
excess NaCl in hyperosmotic environments
(McCormick, 1995). In
Mozambique tilapia, morphological and functional differences between FW and SW
types have been well described in chloride cells of the gills
(Uchida et al., 2000
) and
embryonic yolk-sac membrane (Shiraishi et
al., 1997
; Kaneko et al.,
2002
). In general, SW-type chloride cells are larger than the FW
type and form multicellular complexes together with adjacent accessory cells,
whereas FW-type cells exist individually without forming cellular complexes.
In the present study, gill chloride cells detected with
anti-Na+/K+-ATPase were larger in SW-adapted tilapia
than in FW fish, indicating that FW- and SW-type cells are the predominant
cell types in tilapia adapted to respective environments. Recent studies have
shown that those two types of chloride cells with different ion-transporting
functions can be transformed from one type to another. According to in
vivo sequential observations on chloride cells in the yolk-sac membrane
of tilapia embryos, single FW-type cells are transformed into multicellular
SW-type cells in response to transfer from FW to SW
(Hiroi et al., 1999
).
Similarly, in killifish, SW-type gill chloride cells are transformed into
FW-type cells as a short-term response after transfer from SW to FW
(Katoh and Kaneko, 2003
).
These findings indicate plasticity in the ion-transporting functions of
chloride cells.
It has also been reported that chloride cells are equipped with an
autonomous mechanisms of functional differentiation from the FW to SW type
that are independent of endocrine and nerve systems
(Shiraishi et al., 2001).
Chloride cells in the `yolk ball', a yolk-sac preparation separated from the
body of FW-adapted tilapia embryos, have been shown to form SW-type
multicellular complexes after SW transfer. Considering the functional
plasticity of chloride cells, it is most probable that, in response to
transfer from FW to SW, FW-type cells detect changes in the environmental
salinity in a direct or indirect manner, and this triggers transformation into
the SW type. One possible way is to detect a fluctuation in the external
salinity via the apical membrane of chloride cells facing the
external medium. It is more likely, however, that increased environmental
salinity results in a slight increase in internal osmolality, which could be
detected by the basolateral membrane of chloride cells. In fact, changes in
osmolality on the basolateral side have been shown to affect the rate of
Cl secretion by opercular epithelia of killifish
(Zadunaisky et al., 1995
;
Marshall et al., 2000
).
Based on our observations that AQP3 is intensively located in the
basolateral membrane of chloride cells, we propose a hypothesis that
basolateral AQP3 is involved in osmoreception by chloride cells. It is
expected that the AQP3-rich basolateral membrane is more permeable to water
than the other membranes. This is supported by our finding that AQP3-expressed
Xenopus oocytes exhibited higher water permeability than control
oocytes, resulting in oocyte swelling in the hypo-osmotic medium. Since the
surface area of the basolateral membrane is enlarged because of intensive
infoldings of the tubular system, the ratio of the surface area to the cell
volume is thought to be much higher in chloride cells than in any other cell
type in the gills. Such structural characteristics may further enhance the
possible osmosensitivity of chloride cells. Transfer of fish from SW to FW,
for example, may induce a slight decrease in blood osmolality, which creates
an osmotic gradient between the intracellular fluid and blood. Subsequently,
water moves into chloride cells through basolateral AQP3 according to the
osmotic gradient, resulting in cell swelling. Conversely, transfer from FW to
SW may lead to shrinkage of chloride cells. In mammalian renal cells,
incubation in a hyperosmotic medium decreases cell volume, which leads to
alterations in intracellular ion concentrations and an increase in
Na+/K+-ATPase activity
(Bowen, 1992;
Yordy and Bowen, 1993
).
It is likely that cell volume changes, presumably facilitated by AQP
molecules located in the plasma membrane, may also occur in the other
osmoregulatory organs, the kidney and intestine, in which the gene expression
of AQP3 was confirmed in the present study. In Mozambique tilapia and Japanese
eel, it has been reported that prolactin (PRL)-producing cells in the
pituitary are sensitive to changes in osmotic pressure of the extracellular
fluid. The PRL secretion from organ-cultured pituitaries increased when the
medium osmolality was slightly reduced
(Grau et al., 1981;
Suzuki et al., 1991
), and this
osmosensitive PRL release was linked with volume changes in PRL cells
(Weber et al., 2004
). This
process might also be mediated by AQP3, since we detected substantial
expression of AQP3 in the tilapia pituitary.
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List of abbreviations |
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References |
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