Intestinal water absorption through aquaporin 1 expressed in the apical membrane of mucosal epithelial cells in seawater-adapted Japanese eel
1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life
Sciences, University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
2 Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639,
Japan
3 Japan International Research Center for Agricultural Sciences, Tsukuba,
Ibaraki 305-8686, Japan
* Author for correspondence (e-mail: kaneko{at}marine.fs.a.u-tokyo.ac.jp)
Accepted 2 July 2003
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Summary |
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Key words: Japanese eel, Anguilla japonica, drinking, water absorption, aquaporin 1, intestine
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Introduction |
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Although the water-absorbing mechanism in the intestine of marine or
seawater-adapted fish is still poorly understood, water and ion movements
during the passage of ingested water along the gastrointestinal tract were
well described in early studies (Hickman,
1968; Shehadeh and Gordon,
1969
). In seawater-adapted Japanese eel Anguilla japonica
and European eel A. anguilla, Hirano and Mayer-Gostan
(1976
) have shown that
ingested seawater is mainly desalted in the ion-permeable esophagus, and to a
lesser extent in the stomach, by passive diffusion of the ions into the blood.
The subsequent water absorption is considered to take place in the intestine
following active absorption of monovalent ions. However, the precise mechanism
of water absorption in the intestine is not yet elucidated in eels and other
teleost species.
Aquaporins (AQPs) are a family of integral membrane proteins that function
as water channels, and are importantly involved in fluid transporting
mechanisms in various organs, including the gastrointestinal tract
(Agre et al., 1993;
Ma and Verkman, 1999
;
Matsuzaki et al., 2002
). Many
AQP isoforms have been identified in mammals, and AQP homologues have also
been found in a wide variety of organisms, ranging from bacteria to mammals
(Ishibashi et al., 2000
;
King et al., 2000
;
Verkman, 2002
; Connolly et
al., 1998
; Chispeels and Agre,
1994). In mammalian species, several AQP isoforms are expressed in the
gastrointestinal tract, and are presumably involved in water transport
(Ma and Verkman, 1999
). In
teleost species, however, only three AQP homologues have been identified to
date: AQP0 from the lens of killifish Fundulus heteroclitus
(Virkki et al., 2001
) and AQP3
from the gills of European eel and Japanese dace Tribolodon
hakonensis (Cutler and Cramb,
2002
; Hirata et al.,
2003
). It is not clear, therefore, if AQP is involved in the
water-absorbing process in the fish intestine.
In the present study, to elucidate the mechanisms associated with water absorption in the intestine of seawater-adapted eels, the following experiments were done. (1) To reinvestigate the importance of drinking in seawater-adapted fish, the drinking rate was compared in freshwater- and seawater-adapted eels. (2) Water absorption was examined in intestines isolated from freshwater- and seawater-adapted eels, with reference to the intestinal hydrostatic pressure. (3) A water-channel molecule was explored as a possible water entry site in the intestinal epithelia. We successfully identified homologues of AQP1 from the eel intestine, and compared its expression and morphological distribution in the intestines of freshwater- and seawater-adapted eels.
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Materials and methods |
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Measurement of drinking
To determine the amount of ingested water, freshwater- and seawater-adapted
eels were immersed in 0.01% Trypan Blue in their respective environments. The
fish were sampled at 0, 2, 4, 6 and 18 h after immersion in both experimental
groups. At the time of sampling, the fish were anesthetized with 0.1%
3-aminobenzoic acid ethyl ester (Sigma, St Louis, MO, USA), and the body mass
was determined. The abdominal cavity was opened and the intestine tied off at
the anterior esophagus and the rectum with cotton threads to prevent the
ingested water from leaking. The gastrointestinal tract tied at both ends was
carefully removed, and the ingested water was collected into a conical
graduated measuring container (100 ml). The remaining dye in the tract was
washed out with distilled water, which was combined with the ingested water.
Distilled water was added to the recovered solution to a final volume of 7
ml.
To measure the volume of the ingested water, duplicate sample solutions and
serial dilutions (1, 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64) of the environmental
waters (250 ml each) were placed in wells of a 96-well plate, and the
absorbance at 550 nm measured using a microplate reader (DigiScan 340, Asys
Hitech, Eugendorf, Austria). Since the standard curve was linear within this
range, the dilution factor (DF) was calculated from the regression line. The
amount of ingested water (W; ml kg-1) was calculated from the
following equation:
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Preparation of the intestinal sac
After anesthesia with 0.1% 3-aminobenzoic acid ethyl ester, the intestines
were isolated from freshwater- and seawater-adapted eels. After rinsing out
the contents of the intestines with balanced salt solution (BSS: 140 mmol
l-1 NaCl, 3.0 mmol l-1 KCl, 1.25 mmol l-1
MgSO4, 0.4 mmol l-1 NaH2PO4, 2.0
mmol l-1 NaHCO3, 1.5 mmol l-1
CaCl2, 10 mmol l-1 Hepes, pH 7.4), the intestines were
preincubated in BSS for 1-2 h at 25°C. A polyethylene tube (inner
diameter, 1.4 mm; outer diameter, 1.9 mm), the tip of which was slightly
flamed to make a bulge, was inserted into the anterior end of the intestine,
and fixed with a cotton thread. The bulge prevents the tube from slipping off
the intestine. The other end of the tube was connected to a plastic bottle
containing BSS, which was equipped with a handy air pump and a manometer
(Manostar Gage, 81FN-10E, Okano, Osaka, Japan) so that the pressure could be
regulated. After flushing the air in the lumen with BSS supplied from the
bottle, the posterior end of the intestine was tied with a thread.
The eel intestine could be divided into anterior and posterior parts by its external appearance. In macroscopic observations, the anterior intestine appeared thick and short, while the posterior intestine was thin and long. For the measurement of water absorption in anterior and posterior intestines, sacs were prepared in the same way as described above after separating the intestine into the two parts.
Measurement of water absorption
The intestinal sac was filled with BSS to a hydrostatic pressure of 1.0
kPa, when the length of the intestine between the ties at both ends was
recorded. The pressure was then readjusted to 0.5 kPa, 1.0 kPa or 3.0 kPa, and
the tube was sealed and cut with flamed cutting pliers at a site close to the
anterior end of the intestine. The intestinal sac filled with BSS was lightly
blotted on a Kimwipe, weighed, and incubated in O2-bubbled BSS with
shaking at 25°C. The intestinal sac was weighed to the nearest mg every 30
min for up to 3 h during incubation. The amount of water absorbed across the
intestinal wall was estimated as decrease in mass, normalized to give net
water absorption per cm of the intestine by dividing by the length measured at
1.0 kPa.
Regional differences in water absorption were examined in sacs prepared from the anterior and posterior intestines, filled with BSS to a hydrostatic pressure of 3.0 kPa. The sac loss of mass during an incubation period of 1 h in BSS was determined as described above. The water absorption rate was presented as the mass loss per cm2 of the surface area per hour. For the surface area measurement, the sac was spread serosal side down and the area was determined on the basis that the spread tissue was a rectangle.
Isolation of cDNA fragment encoding AQP
Seawater-adapted eels were used for total RNA preparation. After
anesthesia, the intestines were dissected out, frozen in liquid nitrogen, and
stored at -80°C until RNA extraction. Total RNA was extracted from the
intestines with ISOGEN (Nippon Gene, Tokyo, Japan). Reverse transcription was
carried out using dT-adaptor primer (dT-AP;
5'-ACGGACTCGAGTCGACATCGA(T)17-3') and
SuperscriptTM II reverse transcriptase (Invitrogen, Carlsbad, CA,
USA) at 42°C for 10 min and at 50°C for 1 h.
AQP belongs to the major intrinsic protein (MIP) family, which has two NPA
(Asn-Pro-Ala) motifs that are highly conserved in the most of MIPs from
various animals, plants and bacteria (Park
and Saier, 1996). To obtain cDNA fragments encoding AQP, the
following degenerated primers were designed based on sequences surrounding
these motifs in the MIPs identified so far: sense, fAQP-I
(5'-CAYATHAAYCCIGCNGTNAC-3'); antisense, rAQP-S
(5'-CCRAAISWICKNGCNGGRTT-3'). The first-strand cDNA was amplified
with Taq DNA polymerase (Takara Taq, Takara, Japan) by
polymerase chain reaction (PCR) for 40 cycles (94°C for 30 s, 55°C for
30 s and 72°C for 1 min). The PCR products were ligated into pCR 2.1-TOPO
vector (Invitrogen), and sequenced using a DNA sequencer (SQ-5500, Hitachi,
Tokyo, Japan).
Rapid amplification of cDNA ends (RACE)
For 3' RACE, a gene-specific primer F0
(5'-TCTAGGACACCTAACAGCCAT-3') and adaptor primer 1 (AP1;
5'-ACGGACTCGAGTCGACAT-3') were designed. The reaction product
amplified by 40 cycles of PCR (94°C for 30 s, 60°C for 30 s and
72°C for 1 min) was subcloned into pBluescript SK (-) vector (Stratagene,
La Jolla, CA, USA) and sequenced.
For 5'-RACE, three gene-specific primers, R1 (5'-GCTACGACGTACATGA-3'), R2 (5'-TTGAGCATGCTGATCTGGCA-3') and R3 (5'-CAGCATGCCCAGGGTGA-3') were designed. First-strand cDNA was synthesized from total RNA with R1, as described above. The resulting cDNA was tailed with poly(dA) by a 3'-end labeling kit (Amersham Biosciences, Piscataway, NJ, USA). Using dT-AP, AP1, R2 and poly(dA)-tailed cDNA, the first PCR was conducted in the following profiles: 3 cycles of 94°C for 30 s, 50°C for 1 min and 72°C for 2 min, and 37 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 2 min. The second PCR was performed using AP1, R3 and a tenfold diluted reaction mixture of the first PCR product as a template with a cycle protocol as follows: 40 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min. The second PCR product was subcloned and sequenced as described above.
To confirm the nucleotide sequences of two cDNA clones encoding AQP (S-AQP and L-AQP), 41 cycles of PCR (94°C for 30 s and 68°C for 90 s) were performed with a gene-specific sense primer, AQPexF2 (5'-CAGGGATCCATGACGAAGGAGCTCAAGA-3'), and adaptor primer 2 (AP2; 5'-ACGGACTCGAGTCGACATCGAT-3'). The resultant PCR products containing a full-length open reading frame and 3' untranslated region were ligated into pCR 2.1-TOPO vector and sequenced using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Analysis of the amino acid sequence
Kyte-Doolittle hydropathy profile of the deduced amino acid sequence was
analyzed using GENETIX-MAC version 8.0 software (Software Development, Tokyo,
Japan) at a 10-residue window. The amino acid identities between eel and other
AQPs were analyzed by the BLAST search
(Altschul et al., 1997).
Northern blot analysis
The heart, muscle, liver, intestine, gill, stomach, spleen, swim bladder,
kidney and testis were removed from freshwater- and seawater-adapted eels.
Total RNA was isolated from these tissues, as described above. 20 µg of
total RNA were electrophoresed on a 1.2% formamide-agarose gel and transferred
to BIODYNE B membrane (PALL, East Hills, NY, USA). A cDNA fragment
corresponding to nucleotides 546-975 in S-AQP and to nucleotides 549-978 in
L-AQP was labeled with [-32P]dCTP (Amersham Biosciences)
using MegaprimeTM DNA labeling system (Amersham Biosciences). The
membrane was hybridized with the radiolabeled probe (3.7x105
cpm ml) in 50% formamide, 6x SSC, 2x Denhardt's solution, 0.4%
SDS, and 0.1 mg ml-1 calf thymus DNA at 42°C for 16 h. After
washing in 0.2x SSC containing 0.1% SDS at 60°C for 5 min, the
membrane was exposed to Fuji X-ray film (Fuji Film, Tokyo, Japan). RNA ladder
(Invitrogen) was used as the molecular mass marker.
For quantitative analysis of AQP mRNA, intestines were isolated from
freshwater- and seawater-adapted eels (N=3 each), and cut into the
anterior and posterior parts. RNA extraction and northern blot analysis were
carried out as described above. Hybridization signals were quantified as
described previously (Tsutsui et al.,
2000). The highest mRNA level determined in the posterior
intestine of a seawater eel was designated an arbitrary value of 100.
Antibody
A polyclonal antibody was raised in a rabbit against a synthetic peptide
corresponding to part of the C-terminal region of eel AQP1 molecules (amino
acid residues 241-255 in S-AQP and 242-256 in L-AQP). The antigen designed was
Gly-Pro-Asp-Gly-Asp-Tyr-Asp-Val-Asn-Gly-Pro-Asp-Asp-Val-Pro. 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 (Sawady Technology, Tokyo, Japan).
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 intestine was isolated from
seawater-adapted eel, and spread in lysis buffer consisting of IP buffer (pH
7.4; 140 mmol l-1 NaCl, 2 mmol l-1 KCl, 10 mmol
l-1 Hepes and 5 mmol l-1 EDTA), inhibitors [10 mmol
l-1 Benzamidine, 1 mg ml-1 Pepstatin and 2 mmol
l-1 phenylmethylsulfonyl fluoride (PMSF)] and 1% Triton X-100. The
mucosa and submucosa were scraped off with a blade, homogenized and left in IP
buffer on ice for 40 min to lyse the cells. The lysate was centrifuged at 5000
g for 5 min at 4°C, and supernatant was incubated with 2
ml of the antibody at room temperature for 3 h. Slurry (20 ml)containing 50%
protein A-sepharose beads (Amersham Biosciences, Uppsala, Sweden) was added to
the sample, and the mixture was incubated for 1 h at room temperature. After
washing with IP buffer and centrifugation at 10000 g, 30 ml of
hot Laemmli buffer containing 5% ß-mercaptoethanol was added, and the
mixture was incubated at 65°C for 15 min. The sample was then centrifuged
at 10000 g for 2 min, and the supernatant frozen for later
western blotting.
The sample was separated by SDS-polyacrylamide gel electrophoresis using 7.5% polyacrylamide gels. After electrophoresis, the protein was transferred from the gel to a polyvinylidene difluoride membrane (Atto, Tokyo, Japan). After blocking with 2% skimmed milk at 4°C overnight, the membranes were incubated with the antibody diluted at 1:100 for 1 h at room temperature. The membranes were then incubated with protein A-gold conjugate (British Biocell International, Cardiff, UK) for 1 h and visualized by using silver-enhancing reagents (British Biocell International). The specificity of the immunoreaction was confirmed by incubating the membrane with normal rabbit serum (NRS) in place of the specific antibody.
Immunocytochemistry
To examine the localization of AQP1 and possible differences in its
distribution pattern between freshwater- and seawater-adapted eels, the
intestinal sections were stained immunocytochemically with the antibody
specific for eel AQP1.
After anesthesia, the intestines were removed from freshwater- and seawater-adapted eels (N=3 each). The intestines were washed by flushing with 0.1 mol l-1 phosphate-buffered saline (PBS, pH 7.4), and cut into the anterior and posterior parts. The anterior and posterior intestines were fixed in 4% paraformaldehyde in 0.1 mol l-1 phosphate buffer (pH 7.4) for 18 h, and preserved in 70% ethanol. The intestines were then cut into small pieces (approximately 5 mm) across the axis, dehydrated in ethanol, and embedded in Paraplast. Serial sections were cut at 4 mm thickness and mounted on gelatin-coated slides. To examine the general morphology of the intestines of freshwater- and seawater-adapted eels, some sections were stained with Mayer's Hematoxylin and Eosin.
The sections were immunocytochemically stained using the
avidin-biotin-peroxidase complex (ABC) method
(Hsu et al., 1981), using
commercial reagents (Vectastain ABC kit, Vector Laboratories, Burlingame, CA,
USA). In brief, deparaffined sections were incubated sequentially with: (1)
0.6% H2O2 for 30 min, (2) 2% normal goat serum for 30
min, (3) anti-eel AQP1 diluted at 1:1000 at 4°C, (4) biotinylated
anti-rabbit IgG for 30 min, (5) ABC for 1 h, and (6) 0.02% 3,
3'-diaminobenzidine tetrahydrochloride (DAB) containing 0.005%
H2O2 for 5 min. The sections were observed under a
microscope (Nikon E800, Tokyo) equipped with a differential interference
contrast device. The specificity of the immunoreaction was confirmed by
incubating the sections with NRS in place of the specific antibody.
Statistics
Numerical data were expressed as the mean ± S.E.M.
Statistical analysis was performed using StatView 5.0 software (Hulinks,
Tokyo, Japan). Significant differences at P<0.01 were determined
by Fisher's PLSD after using two-way factorial analysis of variance
(ANOVA).
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Results |
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Water absorption in the intestine
In intestinal sacs prepared from both freshwater- and seawater-adapted
eels, the increase in net water absorption was both time- and hydrostatic
pressure-dependent (Fig. 2).
The loss of sac mass was linear with time for up to 120 min in any
experimental group. The water absorption rate calculated from the linear
regression during the 0-120 min period was greater at higher hydrostatic
pressure (Table 1). Compared at
the same pressure, the water absorption rates were 2.7-3.7 times greater in
seawater sacs than in freshwater sacs.
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|
The water absorption rates tended to be higher in the posterior than in the anterior intestine in both freshwater- and seawater-adapted eels (Fig. 3). The rates were 2.6 times and 3.9 times higher in the posterior than in the anterior in freshwater and seawater eels, respectively. In the posterior intestine, water absorption was significantly greater (P<0.01) in seawater eel than in freshwater eel, whereas the difference was not significant in the anterior intestine.
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cDNA cloning and sequence analysis of AQPs
Based on the DNA sequence of resultant clones, gene-specific primers were
designed for 3' and 5' RACE. The obtained sequences were merged
into a cDNA sequence, which was further confirmed by PCR to generate the
full-length cDNA encoding AQP. As a result, the cDNAs for two AQPs were
isolated, and were designated as S-AQP (DDBJ/EMBL/GenBank accession no.
AB094502) and LAQP (AB094501). S-AQP cDNA consisted of 1195 bp, containing 36
bp of a 5' untranslated region (UTR), 786 bp of an open reading frame
(ORF) and 373 bp of 3' UTR, and LAQP cDNA consisted of 1198 bp,
containing 36 bp of 5' UTR, 789 bp of an ORF and 373 bp of 3'
UTR.
The cDNAs for S-AQP and L-AQP encoded 262 and 263 amino acid residues, respectively. The deduced amino acid sequences of S- and L-AQPs are shown in Fig. 4. Both AQPs possessed the same amino acid sequence, except for a lack of one amino acid in S-AQP (corresponding to Asn116 in L-AQP) and substitutions of two amino acids (Ala117 and Glu125 in S-AQP, Thr118 and Lys126 in L-AQP). According to the hydropathy analysis, both S- and L-AQPs contained six putative transmembrane domains, five connecting loops, and cytoplasmic N- and C-terminal domains, all of which are widely conserved among AQP molecules. The second and fifth loops contained the NPA motifs, which is also characteristic of AQP molecules. Both S- and L-AQPs exhibited the highest identity to mouse AQP1 and frog AQP-channel forming integral protein (CHIP) (59% each). They showed generally high identity to AQP1s in other species (55-59%), whereas the identities to the other AQPs (AQP0 and 2-10) were less than 50%. Thus, S-AQP and L-AQP were considered to be eel homologues of AQP1. Among teleost species, both S-AQP and L-AQP shared 25% identity with European eel AQP3, 48% identity with killifish AQP0, and 24% identity with Japanese dace AQP3.
|
Tissue distribution of AQP1 mRNA
Tissue-specific expression of AQP1 mRNA was analyzed by northern
hybridization (Fig. 5). In both
freshwater and seawater eels, hybridized signals were detected as a single
band of approximately 1.3 kb in the heart, intestine, spleen and swim bladder,
but not in the other tissues examined. It was confirmed that the size of the
mRNA was in agreement with that of the cDNAs for S- and L-AQPs obtained in
this study. The expression levels of AQP1 mRNA in the intestine were higher in
seawater eel than in freshwater eel, whereas such trend was not observed in
the other AQP1-expressing tissues.
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Quantitative analysis of AQP1 mRNA levels in the intestine
The expression levels of AQP1 mRNA in the anterior and posterior intestines
were examined in freshwater and seawater eels
(Fig. 6). Compared separately
in the anterior and posterior intestines, AQP1 mRNA levels were significantly
higher in seawater eel than in freshwater eel (P<0.01). There was
a tendency for AQP mRNA to accumulate more highly in the posterior than in the
anterior in both freshwater and seawater eels; however, the differences were
not significant.
|
Western blot analysis
In western blot analysis, the antibody recognized a major protein band of
approximate size 30 kDa in the intestine of seawater-adapted eel
(Fig. 7). This band was not
detected in the control, in which the membrane was incubated with NRS in place
of the specific antibody. The other bands with higher molecular mass appeared
to be non-specific reactions, and were also observed in the control lane.
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General morphology of the intestine
The intestinal wall consisted of mucosa, submucosa, muscularis and serosa
(Fig. 8). The mucosal
epithelium was composed of a single layer of columnar epithelial cells with a
basally located nucleus. Mucous cells were scattered among the columnar
epithelial cells. In both freshwater- and seawater-adapted eels, mucous cells
were more frequently observed in the posterior intestine than in the anterior.
The muscularis consisted of the inner circular and outer longitudinal muscle
layers. The anterior intestine was generally thicker with the broader
muscularis than the posterior. In the anterior intestine, there was no
structural difference between freshwater and seawater eels. In the posterior
intestine, in contrast, both inner circular and outer longitudinal muscle
layers were thinner in seawater eel than in freshwater eel.
|
Immunocytochemical detection of AQP1 in the intestine
In seawater-adapted eel, the intense immunoreaction for AQP1 was detected
in the apical surface of columnar epithelial cells in the mucosa along the
posterior intestines (Fig.
9A,E). Since the apical membrane of most epithelial cells was
stained with the antibody, the intestinal folds appeared to be lined with the
immunoreactive product, except for the apical surface of mucous cells that
were immunonegative. This was in sharp contrast with the anterior intestine,
in which AQP1 immunoreaction was faint in the apical membrane of mucosal
epithelial cells (Fig. 9C). AQP1 immunoreaction was also faint in the intestine of freshwater-adapted eel
(Fig. 9B,D). In both anterior
and posterior intestines of freshwater eel, not all epithelial cells showed
the immunoreaction, but some cells were immunonegative.
|
In addition to columnar epithelial cells, AQP1 immunoreaction was detected in endothelial cells of the blood vessels distributed in the muscle layers and, to a lesser extent, in the submucosa and mucosa (Fig. 9A). There was no apparent difference in AQP1 immunoreactivity in the endothelia between the anterior and posterior intestines and between freshwater and seawater eels (data not shown).
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Discussion |
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In seawater-adapted eel, ingested water is desalted in the esophagus by
passive diffusion of ions into blood, followed by water absorption in the
intestine (Hirano and Mayer-Gostan,
1976). In the present study, water absorption was much higher in
the intestinal sacs prepared from seawater-adapted eels than in those from
freshwater-adapted eel. Increases in intestinal water absorption have been
reported in eels transferred from freshwater to seawater
(Oide, 1967
; Oide and Utida,
1967
,
1968
;
Kirsch and Meister, 1982
), and
our result is in agreement with those previous reports. Similar profiles have
been observed in coho salmon (Collie and
Bern, 1982
) and Atlantic salmon
(Veillette et al., 1993
)
following parr-smolt transformation.
The intestine of eels is largely divided into two anatomically and
physiologically distinct regions, the anterior and posterior intestines. In
seawater-adapted eel, the present study revealed higher water absorption in
the posterior intestine than in the anterior. Similarly, in seawater-adapted
coho and Atlantic salmon, intestinal water transport has been shown to occur
in the posterior intestine rather than in the anterior
(Collie and Bern, 1982;
Veillette et al., 1993
). The
increased water permeability in the posterior intestine correlated with an
apparent morphological change, whereby the muscle layers became thinner after
seawater transfer (Fig. 8.) In
intestinal sacs from seawater-adapted eel, Ando and Kobayashi
(1978
) showed that the muscle
layers acted as a barrier against water flux, and that the water flux was
increased by stripping off the layers. Thus, the higher water permeability in
the posterior intestine of seawater-adapted eel may be explained at least in
part by the decrease in thickness of the muscle layers.
The decrease in the sac mass was linear with time up to 120 min in any
condition. In seawater-adapted eel, however, the slopes were more or less
decreasing during incubation after 120 min, probably due to the decrease in
the intestinal volume and subsequent decrease in the hydrostatic pressure.
Previous studies have not examined the effect of intestinal hydrostatic
pressure. Collie and Bern
(1982), working with intestinal
sacs from coho salmon, did not find significant differences in loss of sac
mass with time for hydrostatic pressures between 0-20 cm H2O (0-2.0
kPa), which suggested that the initial pressure was not critical for the
determination of the water absorption rate. In the present study, however, the
water absorption rate was apparently greater at higher pressure. Thus, the
initial hydrostatic pressure should be kept constant for the precise
determination and comparison of water absorption rates.
The driving force for active ion transport linked to water absorption in
the intestine is provided at least in part by
Na+/K+-ATPase, since ouabain (a
Na+/K+-ATPase inhibitor) has been shown to inhibit
solute and water transport (Oide,
1967; Collie and Bern,
1982
; Veillette et al.,
1993
). However, although the osmotic gradient established by
active ion absorption drives water uptake in the intestine, the increased
hydrostatic pressure in the intestinal lumen could also contribute to water
transport from the mucosal to serosal side. Although we have not measured the
intestinal hydrostatic pressure in freshwater and seawater eels in this study,
we may speculate that the pressure is higher in seawater fish than in
freshwater fish. The intestines were always more expanded with ingested water
in seawater eel than in freshwater eel. This has also been observed in
freshwater and 80% seawater-transferred medaka larvae, using confocal laser
scanning microscopy in combination with FITC-dextran as an inert marker
(Kaneko and Hasegawa, 1999
).
In mammals, movement of water is the result of the osmotic driving force
created by active salt transport and hydrostatic pressure differences
(Ma and Verkman, 1999
).
Involuntary contractile movements during peristalsis could also increase
intestinal hydrostatic pressure temporally and locally, which would provide an
additional driving force with which to squeeze the water out.
In the present study, we succeeded in cloning two cDNAs encoding AQP from
the eel intestine. AQPs were originally defined as homologue proteins of major
intrinsic protein of mammalian lens that rapidly and selectively permeate
water (Agre et al., 1993). At
least eleven isoforms of AQPs (AQP0-AQP10) have been identified in mammals to
date. S- and L-AQPs represent the first AQP1 homologues identified in teleost
species. Among AQP isoforms, AQPs 1, 2, 4, 5 and 8 are considered to be
water-selective, whereas AQPs 3, 7, 9 and 10 also transport glycerol and other
small solutes (Ishibashi et al.,
1994
,
1997
,
1998
,
2002
;
Echevarría et al.,
1996
). AQP6 has been proposed to function in kidney endosomes as a
pH-sensitive anion channel (Yasui et al.,
1999a
,b
).
Thus, S- and L-AQPs, homologues of mammalian AQP1, are likely to function as
water-specific channels in the intestine.
Amino acid sequences of S- and L-AQPs are the same, except for a lack of one amino acid in S-AQP and substitutions of two amino acids. Because of their minor differences, we performed northern hybridization and immunocytochemical studies without distinguishing the two AQP1 homologues, using probes common to S- and L-AQPs.
Northern hybridization analysis revealed that eel AQP1 was expressed predominantly in the intestine, heart, spleen and swim bladder, but not detected in the other tissues examined. Among the AQP1-expressing tissues, higher expression in seawater eel than in freshwater eel was only seen in the intestine. Further quantitative analysis showed that AQP1 expression levels in the intestine were significantly higher in seawater eel than in freshwater eel, when compared separately in the anterior and posterior intestines. Taken together, these findings indicate that AQP1 expressed in the intestine is importantly involved in water-absorbing processes in the seawater eel intestine.
A specific antibody was raised against eel AQP1. The synthetic peptide corresponding to part of the C-terminal region, common to both S- and L-AQPs, was used as the antigen, so the resulting antibody was considered to react with both molecules. In western blot analysis, the antibody recognized one specific band of approximately 30 kDa, the expected molecular mass of eel AQP1, suggesting high specificity of the antibody.
The immunocytochemical detection of AQP1 in the intestine further supports
the possible involvement of AQP1 in water-absorbing processes in the seawater
eel intestine. In accordance with increased water absorption and predominant
expression of AQP1 in the seawater eel intestine, an intense immunoreaction
for AQP1 was detected in the apical surface of columnar epithelial cells in
the mucosa along the posterior intestine. By contrast, the immunoreaction was
faint in the freshwater eel intestine. Preferential localization of AQP1 in
the apical membrane of epithelial cells in the posterior intestine of seawater
eel indicates that AQP1 may act as a water entry site in the epithelial cells.
It is highly possible that another type of AQP is present in the basolateral
membrane, presumably providing an exit pathway for the release of water to the
serosal side. In the mammalian kidney collecting duct, AQP2 is located in the
apical membrane of the principal cells, whereas AQP3 and AQP4 are present in
the basolateral membrane (Marples et al.,
1999). Such an arrangement of the different AQP isoforms in the
apical and basolateral membranes of the epithelial cells would generally
provide a transcellular pathway for water movement. By contrast, although
several AQP isoforms are expressed in the mammalian gastrointestinal tract,
AQPs have not been localized to the apical membrane of mammalian enterocytes
(Ma and Verkman, 1999
). Thus,
AQPs do not appear to be the primary pathway for water absorption in mammalian
intestines.
In European eel, the expression of AQP3 has been shown in the intestine as
well as in the gills, eye and esophagus
(Cutler and Cramb, 2002). In
contrast with Japanese eel AQP1, European eel AQP3 is not likely to be
responsible for intestinal water absorption. There was no apparent difference
in AQP3 expression between freshwater- and seawater-acclimated eels
(Cutler and Cramb, 2002
), and
AQP3 immunoreactivity was not detected in epithelial cells but in
`intra-epithelial macrophage-like cells' in the intestine
(Lignot et al., 2002
).
In addition to epithelial cells in the intestinal mucosa, AQP1
immunoreaction was observed in endothelial cells of blood vessels distributed
in the muscularis, submucosa and mucosa of the intestine. Similarly, in
mammals, the expression of AQP1 has been observed in microvascular endothelia
of various tissues (Verkman,
2002), including the respiratory system
(Nielsen et al., 1997
) and
kidney (Nielsen et al., 1995
).
Considering that AQP1 expressed in microvascular endothelia has a role in
osmotically driven water transport in mammals, it is most probable that water
absorbed through the intestinal epithelium would then move extracellularly to
blood vessels to flow into the blood circulation.
In the present study, a higher drinking rate was confirmed in seawater-adapted eel than in freshwater-adapted eel. The subsequent water absorption occurred mainly in the posterior intestine. In addition to decreased osmolality of ingested water through active ion absorption, increased hydrostatic pressure may drive water from the mucosal to serosal side through the water-permeable apical membrane of epithelial cells in the posterior intestine of seawater-adapted eel. Increased water permeability of the posterior intestinal wall in seawater eel is attributed to AQP1, which is extensively distributed in the apical membrane of the epithelial cells. The decrease in the thickness of the muscularis would further facilitate the water movement from the epithelial cells in the mucosa to blood vessels in the outer layers in the intestine.
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