Immunolocalisation of aquaporin 3 in the gill and the gastrointestinal tract of the European eel Anguilla anguilla (L.)
School of Biology, University of St Andrews, St Andrews, Fife KY16
9TS, Scotland
1 Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16
9TS, Scotland
2 Gatty Marine Laboratory, University of St Andrews, St Andrews, Fife KY16
9TS, Scotland
* Present address: Université Louis Pasteur, CNRS CEPE, 23 rue Becquerel,
67087 Strasbourg, Cedex 2, France
Author for correspondence (e-mail:
gc{at}st-andrews.ac.uk)
Accepted 7 June 2002
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Summary |
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Key words: aquaporin 3, teleost fish, water channel, urea, European eel, Anguilla anguilla, gill, intestine, immuno-histochemistry, immuno-gold
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Introduction |
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We have recently cloned and sequenced a homologue of the mammalian
aquaporin 3 (AQP-3) from the euryhaline eel Anguilla anguilla L.
(Cutler and Cramb, 2002).
Northern blot analyses indicated that AQP-3 was expressed in the gill,
oesophagus and intestine mainly as a 2.4 kb mRNA species. Quantitative
analysis revealed that the major site of mRNA expression was in the gill of FW
eels and that expression was substantially reduced by up to 94 % following SW
acclimation. In the intestine, the levels of mRNA expression were much lower
and there was no measurable difference between FW and SW-acclimated fish
(Cutler and Cramb, 2002
).
Water channel aquaporins are likely to be central to the molecular and
physiological mechanisms responsible for the changes in water balance that
allow the successful adaptation of euryhaline teleost species to both
freshwater and marine environments. Aquaporins (AQPs) are members of the
ubiquitous family of channel-forming proteins also known as the major
intrinsic protein (MIP) family, and function as water channels that allow
rapid osmotic water flow mainly in epithelial tissues
(Deen and van Os, 1998).
Although AQP isoforms such as AQP-1 are proteins dedicated to water transport,
some isoforms such as AQP-3 also allow the passage of larger polar compounds
such as glycerol and urea (Borgnia et al.,
1999
). In mammals, AQP-3 is located on the basolateral membrane of
renal collecting duct cells, suggesting a role in renal water reabsorption
(Frigeri et al., 1995
). AQP-3
is also present in the airway epithelium
(Frigeri et al., 1995
), eye
conjunctiva and meningeal cells (Lee et
al., 1997
) and is abundant in the nasopharyngeal epithelium,
suggesting a possible role in mucosal fluid excretion and allergic rhinitis
(King et al., 1997
;
Nielsen et al., 1997
). In the
gastrointestinal tract, AQP-3 was detected on the plasma membranes of
stratified squamous epithelial cells in the oesophagus
(Koyama et al., 1999
), along
the basolateral membranes of cardiac gland epithelia in the lower stomach
(Koyama et al., 1999
), in the
columnar epithelia in the villi and crypts of the small intestine from the
jejunum to the ileum and along the basolateral membranes of the columnar
epithelial cells in the colon (Matsuzaki
et al., 1999
; Ramirez-Lorca et
al., 1999
). As indicated by Koyama et al.
(1999
), the presence of AQP-3
in the oesophagus may indicate a role for maintenance of wetness of the
luminal surface of the epithelium. Also, the presence of AQP-3 on the
basolateral membranes of cardiac gland cells in the lower stomach may be
important in cell volume regulation during rapid changes in the osmolality of
the gastric contents (Frigeri et al.,
1995
; Koyama et al.,
1999
).
The aim of this study was to investigate AQP-3 protein expression and to
localise this putative water/urea/glycerol channel within the major
osmoregulatory organs (gill and gastrointestinal tract) of the European eel,
Anguilla anguilla. A double-labelling method was employed using an
eel AQP-3 antibody along with a specific antibody raised against the ß
subunit of the eel Na+,K+-ATPase. Since the
Na+,K+-ATPase is known to be localised within
specialised branchial ion-transporting cells (chloride cells) and intestinal
enterocytes in the eel (Cutler et al.,
2000), the aim of this study was to determine whether AQP-3 is
also colocalised within these cells. Although the movement of salt and water
are inextricably linked, the main route for water transport (either
paracellular or transcellular) has not yet been fully established in either
the intestinal (Ando, 1975
,
1985
;
Alves et al., 1999
) or
branchial (Isaia, 1984
)
epithelium. Experiments were therefore designed to localise the AQP-3 water
channels in both the gill epithelium and the anterior, mid and posterior
regions and rectal segment of the intestine in both FW and SW-acclimated
eels.
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Materials and methods |
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Antibody production
A region of the derived amino acid (aa) sequence of the eel AQP-3, which
was located at the carboxyl end of the sequence and shared the lowest level of
homology with other aquaporins, was chosen for peptide manufacture (aa
915-929; Cutler and Cramb,
2002). An amino-terminal cysteine was added to the peptide for the
purposes of conjugation to the carrier protein. This 16-mer (aa sequence:
CDERIKLSNVATKDAA) was manufactured, coupled to keyhole limpet haemocyanin
(KLH) and used to raise AQP-3-specific polyclonal antisera in rabbits
(Pepceuticals, Ltd, Leicester, UK). A second eel-specific polyclonal antserum
(ß233) raised in sheep against the Na+,K+-ATPase
ß subunit isoform, ß233 (Cutler
et al., 2000
) was also used for labelling and identification of
chloride cells in the branchial epithelium and the enterocytes in the
intestine.
Western blotting
Gill arches and the intestine were quickly removed from FW and 3-week
SW-acclimated eels. Epithelial layers were then scraped free of the underlying
tissue and thoroughly homogenized in 10 vol. (w:v) ice-cold sample buffer [25
mmol l-1 Hepes, 0.25 mol l-1 sucrose, 5 mmol
l-1 MgCl2, 1 mmol l-1 CaCl2, 0.5
mmol l-1 dithiothreitol (DTT), 0.18 mg ml-1 phenylmethyl
sulphonyl fluoride, pH 7.4]. Plasma membrane fractions were isolated by
discontinuous sucrose density gradient centrifugation as described previously
(McCartney and Cramb, 1993).
After centrifugation, membrane fractions banding at the 35-43 % (w:v) sucrose
interface were collected, washed, and finally resuspended in 25 mmol
l-1 Hepes, 0.25 mol l-1 sucrose and 0.5 mmol
l-1 DTT, pH 7.4, before freezing in portions at -25 °C. Protein
concentrations were determined by the method of Bradford
(1976
) and western blotting
was conducted using standard techniques
(Hames, 1996
). In brief,
membrane samples (25 µg protein) were solubilised and denaturated by
incubation at 100 °C for 10 min in a buffer comprising 62.5 mmol
l-1 Tris-HCl, 10 % glycerol, 2 % SDS and 45 mmol l-1
ß-mercaptoethanol, pH 6.8, and denaturated proteins were separated by
SDS-PAGE using 7 % acrylamide gels
(Laemmli, 1970
).
Proteins were electroblotted onto PVDF membranes and immediately processed for immunodetection at room temperature. After blocking the membranes for 1 h with PBS buffer containing 2.5 % BSA (to block non-specific binding sites), the membranes were incubated for 1 h with AQP-3 primary antibody diluted (1:500) in PBS containing 1 % BSA. Control blots were also run simultaneously using equivalent dilutions of either pre-immune serum or immune serum preincubated for 1 h at room temperature with 50 µg ml-1 of the peptide antigen (peptide-negated antiserum). Following washes (3x 15 min) in PBS containing 0.1 % BSA, membranes were incubated for 2 h with an alkaline phosphatase-conjugated secondary antibody diluted 1:3000 in PBS. Bound antibodies were visualized by incubating the blots in a substrate (Western Blue® substrate for alkaline phosphatase, Promega) for 1 min at room temperature. The level of immunoreactivity was then measured as peak intensity (arbitrary units) using an image capture and analysis system (Genesnap/Genetools Image Analysis, Syngene, Cambridge, UK).
Immunofluorescence light microscopy
Gill, intestinal and rectal tissues were fixed for 24 h in either Bouin's
fixative or 4 % paraformaldehyde (PFA). Specimens were fully dehydrated in a
graded ethanol series and embedded in paraffin. Sagittal sections (3 µm)
were cut on a Leitz Wetzlar microtome and collected on poly-L-lysine-coated
slides. The technique for the immunocytochemical identification of AQP-3 in
eel tissues was as described previously
(Lignot et al., 1999).
Sections were preincubated at room temperature for 10 min in 0.01 mmol
l-1 Tween 20, 150 mmol l-1 NaCl in 10 mmol
l-1 phosphate buffer, pH 7.3, and then treated with 50 mmol
l-1 NH4Cl in PBS, pH 7.3, for 5 min to reduce background
associated with free aldehyde groups of the fixative. The sections were washed
in PBS (1x 5 min) and incubated for 10 min with a blocking solution (BS)
containing 1 % bovine serum albumin (BSA) and 0.1 % gelatin in PBS. Droplets
(10 µl) of primary antibody diluted (1:100) in BS were placed on the
sections and incubated for 1 h at room temperature in a wet chamber. After
being washed in BS (6x 5 min), the sections were incubated for 2 h in
droplets of secondary antibody [1:200; FITC-conjugated donkey anti-rabbit
IgGH&L (Jackson ImmunoResearch)]. Following extensive washes in
BS (3x 5 min) and in PBS (3x 5 min), sections were mounted with
anti-bleaching mounting medium (Sigma). Sections were then examined with a
fluorescence microscope (Leitz Dialux 20 coupled to a Ploemopak 1-Lambda lamp)
equipped with the appropriate filter set (450-490 nm band-pass excitation
filter) and a phase-contrast device. The procedure was similar for the control
sections, which were incubated with the pre-immune serum at the same dilution
as for the primary antibody.
Confocal laser scanning microscopy
Specimen preparation was similar to the one used for the immunofluorescence
light microscopy (see above). Branchial, intestinal and rectal sections were
then incubated for 1 h at room temperature in a wet chamber in droplets
containing both the AQP-3 antibody raised in rabbit and the eel ß233
antibody raised in sheep, each at its optimal dilution in BS (1/100 and 1/50,
respectively). The ß233 antibody was used as a cellular marker as it
specifically labels the ß subunit of the
Na+,K+-ATPase that is expressed at high levels in gill
chloride cells and intestinal enterocytes
(Cutler et al., 2000). After
being washed in BS (6x 5 min), the sections were incubated for 2 h in
droplets of mixed FITC- and Cy3-conjugated secondary antibodies [1:200; donkey
anti-rabbit IgGH&L and 1:200; donkey anti-sheep
IgGH&L (Jackson ImmunoResearch)]. Following washes in BS
(3x 5 min) and then in PBS (3x 5 min), sections were mounted with
anti-bleaching mounting medium (Sigma). Appropriate controls indicating no
cross-reaction of the secondary antibodies were carried out in parallel.
A confocal laser scanning microscope (Biorad MRC 600) equipped with a krypton/argon laser and a Nikon Diaphot microscope (objectives: 20x, 0.75 numerical aperture and 60x, 1.4 numerical aperture, oil immersion) was used in combination with excitation by blue light (488 nm), a 515 nm emission barrier filter (BHS) and an A2 filter (blocking emission wavelengths below 600 nm). With this set-up the emission wavelengths of Cy3- and FITC-conjugate are separated and transmitted to different photomultipliers. The pictures from each photomultiplier were subsequently merged in false colour to visualise the labels simultaneously (green colour: FITC-conjugates; red colour: Cy3-conjugates).
Transmission electron microscopy
Gill samples from SW-acclimated eels were fixed on ice for 1-2 h with 2.5 %
glutaraldehyde in 0.1 mol l-1 sodium cacodylate buffer, pH 7.5.
They were post-fixed in 1 % osmium tetroxide in 0.1 mol l-1 sodium
cacodylate buffer, pH 7.5, for 1 h at 4 °C, fully dehydrated in a graded
ethanol series and infiltrated with LR-White (Agar Scientific), which was
polymerised for 24 h at 60 °C. Ultrathin sections were cut on a Leica
ultramicrotome and collected on Formvar-coated nickel grids. Sections were
stained with 1 % uranyl acetate (30 min) and Reynolds lead citrate (3 min) and
observed on a Philips EM 301 transmission electron microscope.
Immuno-gold electron microscopy
A post-embedding immunostaining technique on LR White sections was applied,
as previously described (Lignot et al.,
1999). Gill samples from SW-acclimated silver eels were fixed for
1-2 h with 0.5 % glutaraldehyde in 0.1 mol l-1 sodium cacodylate
buffer, pH 7.5, adjusted to plasma osmolality (390 mOsm kg-1) by
addition of NaCl to avoid osmotic shock, fully dehydrated in ethanol,
infiltrated with LR-White, and polymerized for 18 h at 50 °C. Ultrathin
sections were cut on a Reichert TM60 ultramicrotome and collected on
Formvarcoated nickel grids. Selected grids were placed on 5 % gelatin
containing 50 mmol l-1 glycine, pH 7.3. The grids were successively
preincubated on droplets of 50 mmol l-1 glycine in PBS (1x 5
min) and 1 % BSA-PBS (3x 5 min). The grids were then transferred to
droplets of AQP-3 antibody diluted to 1:25 with BSA-PBS and incubated for 2 h
at room temperature in a wet chamber. The sections were washed in BSA-PBS
(6x 5 min) and incubated for 1 h in droplets of 15 nm gold-conjugated
goat anti-rabbit IgG (Jackson ImmunoResearch). After washing in 1 % BSA-PBS
(3x 5 min) and PBS (3x 5 min), sections were stained with 1 %
uranyl acetate (30 min) and Reynolds lead citrate (3 min) and studied using a
Philips EM 301 electron microscope. For the controls, the procedure was
similar but the grids were incubated with the pre-immune serum.
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Results |
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Immunofluorescence light microscopy
Bouin and/or 4 % PFA fixation and paraffin embedding procedures yielded
good antigenicity and good structural preservation (Figs
3,
4). In the gill, although
AQP-3-specific staining was apparent throughout the epithelium, the chloride
cells within the primary filaments and at the proximal ends of secondary
lamellae in SW-acclimated eels (Fig.
3A,B) and along both the primary filaments and secondary lamellae
in FW fish, exhibited much stronger immunoreactivity
(Fig. 3D,E). Although chloride
cells were present throughout the branchial epithelium, a much higher cell
density, and therefore immuno-positive staining, was found near the trailing
edge of the filaments (results not shown). Immunoreactivity was present
throughout the entire chloride cell although heavier staining was always
noticeable towards the apical surface (Fig.
3A,B,D,E). A much lighter general staining was also observed
throughout the other epithelial cells where immunoreactivity was predominantly
located towards the cell periphery, indicating that AQP-3 was mainly localised
on or very close to the plasma membrane. In FW eels, cells near the central
cavity of the primary filament epithelium
(Fig. 3G) and basal layer cells
within the gill arch epithelium (Fig.
3H) exhibited strong immunoreactivity near the plasma membrane.
This staining was present but not as intense in the SW-acclimated group of
fish (results not shown). In both FW and SW-acclimated fish, controls using
pre-immune serum showed no positive immunoreactivity with only non-specific
auto-fluoresence being restricted to red blood cells
(Fig. 3C,F).
|
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In the intestinal epithelium of FW and SW-acclimated eels, neither the numerous goblet cells, nor the columnar enterocytes with their apical brush border, stained with the AQP-3 antibody (Fig. 4A,B). Immunoreactivity was only found in large macrophage-like bodies found throughout the entire length of the intestinal epithelium of FW silver eels and primarily within the anterior intestine of SW-acclimated eels (Fig. 4A,B). The precise nature of these cells awaits further investigation. In the rectal epithelium of SW-acclimated eels, copious staining was also observed within the goblet cells (Fig. 4D). In FW eels, however, only a few goblet cells presented positive staining (Fig. 4E). In both FW and SW-acclimated fish, control pre-immune serum showed no positive immunofluorescence within the digestive tract, with only non-specific auto-fluorescence again restricted to red blood cells (Fig. 4C,F).
Confocal laser scanning microscopy (CLSM)
Fig. 5 shows CLSM scans of
the double-labelled gill, intestinal and rectal epithelia of FW and
SW-acclimated eels. Intense staining, from both ß233 and AQP-3
antibodies, was restricted to the chloride cells of FW and SW-acclimated eels
(Fig. 5A-D). Staining
corresponding to the ß233 subunit of the
Na+,K+-ATPase was restricted to the region containing
the basolateral tubular network of the chloride cell (see below) but was much
reduced towards the apical surface and completely absent around the apical pit
region (Fig. 5A-D). In
contrast, however, the AQP-3 antibody exhibited immunoreactivity throughout
most of the chloride cell with increased intensity near the apical pit region,
where ß233 immunoreactivity was absent
(Fig. 5B-D). In the intestine
of both FW and SW-acclimated eels, ß233 immunoreactivity was observed
along the cell surface of the enterocytes
(Fig. 5E,F) while AQP-3
immunoreactivity was restricted to large macrophage-like cells, which were
randomly distributed within the columnar epithelium
(Fig. 5E,F). Finally, in the
rectum of SW-acclimated eels, while the ß233 was detected within the
enterocytes, AQP-3 was almost entirely confined to the goblet cells
(Fig. 5G).
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Transmission and immuno-gold electron microscopy
In gill chloride cells from the SW-acclimated eel, mitochondria are evenly
distributed throughout the cell except for the area around the apical pit
(Fig. 6A,B). An extensive
smooth-surfaced tubular system is juxtaposed to the mitochondria
(Fig. 6A). This tubular system
forms a network from the basal to the apical part of the cell. Just below the
flat or rounded apical pit, numerous small densely packed vesicles form a
tubulovesicular system underneath the apical membrane
(Fig. 6B).
|
On gold labelled sections, gold particles were predominantly localized in chloride cells within membranes of this apical tubulovesicular system and in the subjacent basolateral tubular network (Fig. 6C, 6D). Control sections using preimmune serum exhibited only low levels of non-specific staining (Fig. 6E).
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Discussion |
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The leakiness of the branchial epithelium is considered to account for over
90 % of the total body water influx in FW fish
(Motais et al., 1969;
Haywood et al., 1977
).
Although the diffusional water permeability is not radically different between
FW and SW-acclimated euryhaline teleosts, the osmotic water permeability is
generally higher in FW fish (Isaia,
1984
). The gut of marine teleosts also plays an essential part in
compensating for the osmotic water loss through the gills, the oesophagus and
anterior intestine being the main areas in processing the ingested seawater
(Kirsch and Meister, 1982
).
Seawater is processed along the gut in two steps: essentially ion diffusion
with little net water uptake across the oesophagus
(Kirsch and Laurent, 1975
;
Hirano and Mayer-Gostan, 1976
;
Parmelee and Renfro, 1983
;
Simmonneaux et al., 1988
),
followed by active NaCl transport coupled to water absorption in the intestine
(House and Green, 1965
;
Skadhauge, 1969
,
1974
;
Field et al., 1978
;
Frizzell et al., 1984
).
Aquaporin 3 (AQP-3), known to be a key water channel protein in mammals,
has been identified in both FW and SW-acclimated silver eels using a specific
polyclonal antibody directed against the C-terminal of the eel protein.
Western blotting has revealed that the AQP-3 protein is predominantly
expressed within the gill epithelium. SW acclimation resulted in a reduction
of approximately 65 % in the expression of the branchial protein, suggesting
that AQP-3 may play an important functional role in this osmoregulatory organ.
Immunohistochemical studies indicated a qualitatively similar amount of AQP-3
staining within chloride cells from both FW and SW-acclimated fish. However,
the results clearly demonstrated that gills from FW fish exhibited elevated
expression of the protein in epithelial cells deep within the primary
filaments and near the branchial arch, where the epithelial layers appeared
much thicker than in the SW-acclimated fish. This staining predominated close
to the plasma membrane of the cells and particularly on the serosal side of
the epithelium. This staining, which was greatly reduced within the gills of
SW-acclimated eels, could at least partially explain the marked downregulation
of AQP-3 protein expression following SW transfer. These localisation studies
also suggest that water movement via AQP-3 may occur across the
serosal membrane into the epithelial cells in order to protect the cells from
dehydration. Such a role for APQ-3 in fish epithelium could therefore be
similar to that suggested for the same protein that is expressed in rat skin
and urinary bladder epithelia (Matsuzaki et al.,
1999,
2000
). This contention is
further strengthened by reports that AQP-3 expression can be upregulated by
increasing the osmolality of the extracellular medium
(Matsuzaki et al., 2001
).
AQP-3 water channel expression was also observed within the chloride cells of
both FW and SW-acclimated eels, where it was co-expressed along with the
ß233-isoform of the Na+,K+-ATPase. AQP-3
immunoreactivity was also abundant towards the apical surface and particularly
around the apical pit of the chloride cells, where
Na+,K+-ATPase is absent. This apical location for AQP-3
in the chloride cells was unexpected, as in mammals AQP-3 is mainly expressed
in the basolateral membranes of both the renal collecting duct cells and of
epithelial cells lining the villus tip of the small intestine and colon
(Frigeri et al., 1995
;
Ramirez-Lorca et al., 1999
;
Koyama et al., 1999
).
The presence of high levels of expression of AQP-3 in the chloride cells
correlates with physiological and morphological evidence indicating that these
cells provide a major route for water as well as ion movement in SW
(Isaia, 1984;
Ogasawara and Hirano, 1984
).
The immunohistochemical localisation of AQP-3 suggests a possible association
with the basolateral tubular network of the chloride cells, and this
could be related to the osmotic water flux pathway that has been hypothesised
to be operating in the system (Isaia,
1984
). The possible location of AQP-3 within the
tubulovesicular system surrounding the apical pit region also suggests
that another regulated water pathway may occur within the chloride cells of
both FW and SW-acclimated fish. The tubulovesicular system situated
between the apical plasma membrane of the chloride cells and the tubular
reticulum has already been hypothesized as a transient communication channel
between the internal and external milieu
(Sardet et al., 1979
).
Another potential physiological role for AQP-3 in the branchial chloride
cells is in the excretion of nitrogenous waste products. Many studies have
shown that the AQP-3 isoform can be associated with the transport of small
polar solutes such as urea and glycerol
(Deen and van Os, 1998;
Borgnia et al., 1999
;
Verkman and Mitra, 2000
). In
teleost fish, nitrogenous waste products such as urea and ammonia can be
excreted by extra-renal routes, including the gill
(Masoni and Payan, 1974
;
Isaia, 1984
). In the
ammoniotelic European eel, branchial and renal excretion of urea occur at the
same rate and branchial urea excretion is threefold lower in SW compared to
FW-acclimated eels (Masoni and Payan,
1974
). Branchial urea clearance in ureotelic fish species is
believed to take place through vesicular trafficking in pavement cells
(Laurent et al., 2001
).
Ammoniotelic teleost fish, however, do not show such vesicular trafficking
(Laurent et al., 2001
), with
branchial urea clearance believed to take place through the chloride cells
(Masoni and Garcia-Romeu,
1972
). The presence of AQP-3 in the basolateral network and
within the apical vesiculotubular network of the chloride cells could
therefore be related to the clearance of this nitrogenous waste product.
In the eel intestine, increased water absorption is observed after
increasing the external salinity (Maetz
and Skadhauge, 1968). Considerable ingestion of seawater occurs at
the moment of transfer (Kirsch,
1972
; Kirsch and Mayer-Gostan,
1973
), which is suggestive of a drinking reflex that is possibly
linked to the increase in chloride concentration and/or associated local
cellular dehydration within the buccal cavity
(Hirano, 1974
;
Ando and Nagashima, 1996
). The
osmotic water permeability of the intestine increases by two- to sixfold
following SW transfer, with the highest water fluxes occurring across the mid
region of the gut followed by the posterior, anterior and rectal regions,
respectively (Ando and Kobayashi,
1978
; Ando, 1980
).
The very low levels of AQP-3 protein expression observed in western blots when
using intestinal extracts from both FW and SW-acclimated eels correlates with
the relatively low AQP-3 mRNA expression observed in this tissue
(Cutler and Cramb, 2002
).
Expression of mRNA and protein within the intestine is mainly associated with
the positive immunoreactivity discretely localised within intra-epithelial
macrophages, which were distributed throughout the intestinal epithelia, and
to the goblet cells, which were mainly located near the rectum. The lack of
AQP-3 expression in the eel intestinal columnar cells, however, agrees with
recent studies which suggested that there was an absence of water channels in
eel brush border membrane vesicles (Alves
et al., 1999
). However, it is highly likely that other AQP
homologues are present within the teleost intestinal epithelia. In mammals a
number of aquaporin isoforms, including AQP-1, AQP-4, AQP-7 and AQP-8, have
been characterised in the small intestine (Nielsen et al., 1993;
Kuriyama et al., 1997
;
Koyama et al., 1999
;
Ma and Verkman, 1999
;
Elkjaer et al., 2001
).
Finally, the strong expression of AQP-3 protein in the rectal goblet cells of
SW-acclimated eels suggests a major role for this protein in water trafficking
associated with mucus secretion. A role for maintenance of `wetness' on the
luminal surface of the rectal epithelium can also be hypothesised, as
speculated for AQP-3 in the rat oesophagus
(Koyama et al., 1999
) and for
AQP-5 in corneal squamous epithelial cells in the eye
(Funaki et al., 1998
).
In conclusion, this study indicates that the AQP-3 protein is expressed in osmoregulatory tissues of teleost fish. The cellular localisation in branchial chloride cells indicates that AQP-3 could be involved in water transport and/or with nitrogenous waste excretion hypothesised to be operating in the basolateral tubular and apical vesiculotubular systems of chloride cells. The presence of AQP-3 in the basal cells of the gill arch epithelium and of the gill primary filaments of FW-acclimated eels also suggests regulated water movement within or across other epithelial cells, particularly those associated with the serosal side of the gill epithelium. AQP-3, however, is unlikely to play any major role in the intestinal water absorption that occurs following the drinking response in SW-acclimated fish. Furthermore, the expression of AQP-3 within the rectal mucus cells may be associated with the maintenance of mucus layer fluidity in the rectum.
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Acknowledgments |
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References |
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