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First published online June 26, 2009
Journal of Experimental Biology 212, 2183-2193 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.029322
African lungfish, Protopterus annectens, possess an arginine vasotocin receptor homologous to the tetrapod V2-type receptor
1 Department of Biological Science, Graduate School of Science and Engineering,
University of Toyama, Toyama, Japan
2 Laboratory of Physiology, Ocean Research Institute, University of Tokyo,
Tokyo, Japan
3 Department of Biochemistry, National Cardiovascular Center Research Institute,
Osaka, Japan
* Author for correspondence (e-mail: uchiyama{at}sci.u-toyama.ac.jp)
Accepted 2 April 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: arginine vasotocin (AVT), V2-type receptor, kidney, osmoregulation, lungfish
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). In tetrapods,
the antidiuretic action of AVP and AVT are mediated through two subtypes of
AVP/AVT receptors, namely V1a- (Morel et
al., 1992) and V2-type receptors
(Lolait et al., 1992). The
V1a-type receptors are linked intracellularly to the phospholipase C
(PLC)/protein kinase C (PKC) signaling pathway and exert an antidiuretic
action by regulating vascular smooth muscle contraction and thereby modifying
the glomerular filtration rate (Bankir,
2001). By contrast, the responses through the V2-type receptor are
mediated by the adenylyl cyclase (AC)/protein kinase A (PKA) signaling system
(Thibonnier et al., 1998), and
the V2-type receptor is involved in water reabsorption by promoting the
expression and insertion of the AVP/AVT-dependent water channel aquaporin 2
(AQP2) in the luminal membrane of the distal convoluted tubule and the
collecting tubule in the kidney (Lolait et
al., 1992; Nielsen et al.,
2002).
All tetrapods possess two types of renal water conservation mechanisms
through the V1a- and V2-type receptors; however, only the V1a-type receptor
and its vascular effects have been reported in fish
(Mahlmann et al., 1994;
Warne, 2001). Sawyer and
colleagues reported that in the South American lungfish, Lepidosiren
paradoxa, the effects of exogenous AVT appear to be primarily responsible
for vasoconstriction and have no effect on tubular antidiuresis
(Sawyer et al., 1982).
Therefore, the tubular antidiuretic action through the V2-type receptor was
thought to have emerged in the terrestrial tetrapods
(Pang, 1983). However, Balment
and colleagues suggested that the AVT receptor associated with the AC/cAMP
signaling pathway might be present in teleosts
(Perrott et al., 1993;
Warne, 2002). However,
molecular biological evidence of the V2-type receptor has not been reported
thus far in fish.
Lungfish, recognized as living fossils, are an archaic group of fish belonging to the class of lobe-finned fish (Sarcopterygii), which differ from the class of ray-finned fish (Actinopterygii). African lungfish, including Protopterus aethiopicus, Protopterus annectens and Protopterus dolloi, spend an aquatic life in flooded swamplands in the rainy season but can estivate in subterranean mud cocoons without water during the dry season. Thus, during the switch from freshwater (FW) to estivating status, they experience opposite changes in body fluid balance. In the FW lungfish, the main physiological function of the kidney is to excrete excessive water. By contrast, during estivation (EST), the lungfish must retain water in the body, enabling it to survive for months without water. Thus, we suppose that African lungfish employ two types of water conservation mechanisms through vascular V1a-type and tubular V2-type receptors in the kidney.
It is interesting to identify AVT receptors expressed in the kidney of lungfish because lungfish might be a key species for the investigation of the molecular and functional evolution of the neurohypophysial hormone system in vertebrates, in particular from aquatic fish to terrestrial tetrapods. Here, we report that the AVT receptor, homologous to the tetrapod V2-type receptor, is expressed in the kidney of the African lungfish, P. annectens. The functional significance of the lungfish renal V2-type receptor will be discussed in terms of the ecological characteristics, i.e. estivation in a dry environment.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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80% humidity in plastic bags (12 cmx12 cmx30 cm,
LxWxH) containing a mass of damp cotton. It took 1 week for the
lungfish to be encased in a brown dried mucus cocoon, and the lungfish (EST;
N=9) were allowed to estivate for 90 days. Specimens in freshwater
(FW; control; N=9) were placed in dechlorinated tap water for the
same period of time. After 90 days, the FW specimens were anesthetized with
0.1% ethyl 3-aminobenzoate methanesulfonate (Sigma, St Louis, MO, USA), while
the EST specimens were killed directly by pithing. Blood samples were
collected by cardiac puncture using heparinized 1-ml syringes and hematocrit
capillaries for determination of the plasma components. The tissue samples
were quickly dissected and frozen in liquid nitrogen. The experiments
described in this manuscript were performed according to the Guidelines for
Care and Use of Animals approved by the ethics committees of the University of
Toyama and the University of Tokyo.
Analyses of plasma components
Plasma osmolality and plasma sodium concentrations were measured using an
osmometer (Wescor 5520, Logan, UT, USA) and an atomic absorption
spectrophotometer (Hitachi Z5300, Tokyo, Japan), respectively. Plasma urea
concentration was measured by using the Wako Urea NB test (Wako Pure Chemical,
Tokyo, Japan) in vitro enzymatic colorimetric method.
cDNA cloning
Total RNA was extracted from the kidney of the FW specimens using the
Isogen reagent (Nippon Gene, Tokyo, Japan). First-strand kidney cDNA was
reverse-transcribed from DNase I-treated total RNA using the
PrimeScriptTM 1st strand cDNA Synthesis Kit (TaKaRa Bio, Otsu, Shiga,
Japan). Degenerate primers (Table
1) for AVT receptors were designed based on the alignment of the
three subtypes (V1a, V2, V1b/V3) of the mammalian AVP receptors
(Lolait et al., 1992;
Morel et al., 1992;
Sugimoto et al., 1994),
amphibian AVT receptors (Kohno et al.,
2003; Acharjee et al.,
2004; Hasunuma et al.,
2007) and teleost V1a-type receptors
(Mahlmann et al., 1994;
Warne, 2001). Polymerase chain
reaction (PCR) was performed using BIOTAQ DNA polymerase (Bioline, London, UK)
as follows: 94°C for 2 min, 35 cycles at 94°C for 1 min, 55°C for
30 s, 72°C for 40 s, and finally 72°C for 10 min. The PCR products
were separated by electrophoresis, and the major band of the predicted size
was purified from the sliced gel and ligated into the pT7Blue T-Vector
(Novagen, San Diego, CA, USA). The ligated plasmid was transformed into the
competent cell (XL1-Blue, Invitrogen, Carlsbad, CA, USA). Plasmid DNA was
isolated by a modified alkaline/SDS method (Rapid Plasmid Purification
Systems, Marligen Bioscience, Ijamsville, MD, USA). The sequencing reaction
was performed using the BigDye Terminator Cycle Sequencing Kit (Applied
Biosystems, Foster City, CA, USA). The nucleotide sequence was determined
using an ABI PRISM 310 or 3100 DNA sequencer (Applied Biosystems). Full-length
cDNAs were obtained by 5'- or 3'-rapid amplification of the cDNA
ends (RACE) using adaptor and gene-specific primers
(Table 1).
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Molecular phylogenetic analysis
The deduced amino acid sequences of the lungfish AVT receptors were aligned
with those of the other neurohypophysial hormone receptors using ClustalX
version 1.83 (Thompson et al.,
1997). The aligned amino acid sequences were analyzed by the
neighbor-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML)
methods. The NJ and MP analyses were conducted using MEGA4 software
(Tamura et al., 2007), and the
ML analysis was conducted using the Bio Edit program version 7.08 (Ibis
Biosciences, Carlsbad, CA, USA). To estimate the reliability of the trees,
bootstrapping of the data (1000 replicates) was performed.
Functional analyses of the cloned receptors
Intracellular accumulation of Ca2+ and cAMP was analyzed to
determine the intracellular signaling system of the cloned receptors.
Functional analyses of cloned receptors were conducted using Chinese hamster
ovary (CHO) cells transiently expressing lungfish V1a-type or V2-type
receptor. The CHO cells were cultured in alpha-MEM (Gibco, Invitrogen)
containing 10% fetal calf serum (FCS) at a density of 1x106
cells in a 10-cm dish for 24 h. Expression vectors (pcDNA3.1/V5-His TOPO;
Invitrogen) containing the coding region of lungfish V1a-type or V2-type
receptor (2.5 µg) were transfected with FuGENE6 (Roche Diagnostics)
according to the manufacturer's protocol. Twenty-four hours after
transfection, the transfected CHO cells were plated onto a normal 96-well
black plate at a density of 3x104 cells per well. The
measurement of intracellular Ca2+ mobilization was performed using
FLIPRtetra (Molecular Devices, Menlo Park, CA, USA). Twenty hours after
plating, the culture medium was aspirated, and 100µl fluorescent dye
solution containing 4.4 µmol l–1 Fluo-4AM (Invitrogen), 1%
FCS and 0.045% pluronic acid (Sigma) in a working buffer [1xHank's
Balanced Salt Solution (Gibco):20 mmol l–1 Hepes buffer
containing 250µmol l–1 probenecid (Sigma)] was loaded onto
each well. The plate was incubated for 1 h at 37°C in a CO2
incubator or at room temperature. After washing three times with the working
buffer, 100µl of AVT and AVP (Peptide Institute, Osaka, Japan) in a working
buffer containing 0.001% Triton X-100 were automatically added to the well by
the FLIPR system. Intracellular Ca2+ changes were measured by
excitation at 488 nm and emission at 500–560 nm.
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RT-PCR
The tissue distribution of lungfish V1a- and V2-type receptor mRNAs was
examined by RT-PCR. Total RNA was extracted from various tissues (brain, eye,
internal gill, lung, heart, liver, gall bladder, pancreas, intestine, kidney,
ovary, testis, muscle and ventral skin) of the FW and EST specimens using the
Isogen reagent (Nippon Gene). After treatment with DNase I (Invitrogen) to
remove the genomic DNA, cDNA was synthesized from 1 µg of total RNA using
the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics). PCR was
performed using specific primers (Table
1) for individual genes [V1a-type receptor, V2-type receptor and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. GAPDH mRNA cloned from
P. annectens (DDBJ accession no. AB426480) was used as an internal
standard to estimate the relative levels of objective mRNA expression. The PCR
conditions comprised 34 cycles (V1a-type receptor), 32 cycles (V2-type
receptor) and 24 cycles (GAPDH) of 40 s at 94°C (denaturation), 30 s at
55°C (annealing) and 40 s at 72°C (extension) in 20-µl reaction
mixtures.
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Antibodies
A polyclonal antibody against lungfish V2-type receptor was raised by
immunizing Japanese white rabbits with the synthetic peptide
NH2-CVHNKFRRKSIGEDS-COOH, corresponding to amino acid residues 345–359
of lungfish V2-type receptor. The antiserum was collected and purified using
an affinity column bearing the immobilized synthetic peptide with affinity gel
beads (Affi-Gel 10; Bio-Rad, Hercules, CA, USA). Anti-vacuolar type
H+-ATPase 
-subunit antiserum was raised by immunizing
Japanese white rabbits with a synthetic peptide,
NH2-AEMPADSGYPAYLGAR-COOH, as described previously
(Hayashi et al., 2000). This
antiserum specifically recognizes intercalated cells of the late distal tubule
and the collecting duct of the amphibian kidney
(Uchiyama and Yoshizawa, 2002;
Konno et al., 2006;
Konno et al., 2007;
Kumano et al., 2008).
Western blotting
Tissue samples from the kidney, heart and liver were homogenized in
transmembrane protein extraction buffer I (ProteoExtract Transmembrane Protein
Extraction Kit; Novagen) using a tissue homogenizer (Physcotron NS-310E,
Nition, Chiba, Japan). The homogenates were centrifuged at 1000
g for 5 min at 4°C to collect the insoluble pellets
containing the cell membrane. The pellets were solubilized in extraction
buffer II (ProteoExtract Transmembrane Protein Extraction Kit; Novagen) and
were then centrifuged at 16,000 g for 15 min at 4°C to
collect the membrane fraction containing the transmembrane proteins. The total
protein concentration of the membrane protein fractions was measured by the
Bradford method (Bradford,
1976) using a Protein Assay Reagent (Bio-Rad). Samples containing
40 µg of transmembrane proteins were degenerated at 60°C for 15 min in
Laemmli buffer, separated by SDS-PAGE using 10% polyacrylamide gel and then
transferred from the gel to a nitrocellulose membrane (Hybond-C; GE Healthcare
Bio-Sciences, Piscataway, NJ, USA). To prevent non-specific binding, the
blotted membranes were blocked with 5% skimmed milk for 2 h at room
temperature and were then probed overnight at 4°C with the lungfish
V2-type receptor antibody (dilution 1:2000 with 1% BSA-PBS). After washing
with TBS-Tween 20, the membranes were incubated with HRP-conjugated
anti-rabbit IgG (ECL plus Western Blotting Detection System; GE Healthcare
Bio-Sciences) for 2 h at room temperature. After further washing of the
membranes with TBS-Tween 20, immunodetection was performed by using an
enhanced chemiluminescence kit (ECL plus Western Blotting Detection System).
Autoradiographs were obtained by exposure to X-ray films (Hyperfilm ECL; GE
Healthcare Bio-Sciences). As a control, the primary antibody was replaced with
the lungfish V2-type receptor antibody preincubated with 1 µg
ml–1 of the immunogen peptide.
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-subunit antibody (dilution 1:4000 with PBS) for
24 h at 4°C. After rinsing with PBS, the sections were incubated for 2 h
at room temperature with biotinylated swine anti-rabbit IgG and then with the
avidin-biotin-peroxidase complex. After rinsing with PBS, the immunoreactivity
was visualized with 3'-diaminobenzidine solution (Sigma) containing
0.01% H2O2. Immunolabeling control for the lungfish
V2-type receptor and vacuolar-type H+-ATPase was performed by
preabsorption of primary antibodies with excess peptides of each immunogen (1
µg ml–1). All control experiments were negative for
immunostaining.
Statistics
Values are expressed as means ± s.e.m. (standard error of the mean).
To test the difference between the groups, two-tailed, paired and unpaired,
Student's t-tests were used in the present study. Statistical
significance was established at P<0.05 and P<0.01.
Correlation was calculated by Spearman's correlation analysis. P
values less than 0.01 were considered to be statistically significant.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Molecular trees were constructed using the amino acid sequences of the cloned receptors and other neurohypophysial hormone receptors. Since three different methods yielded the same tree, the tree inferred by the NJ method is shown in Fig. 3. Snail conopressin receptor (CPR) was used as the out-group. The receptors were classified into four groups, namely V1a-type, V1b-type, V2-type and oxytocin receptor. The two types of AVT receptors cloned from lungfish kidney were classified into the V1a- and V2-type receptor subtypes. In each subfamily, the lungfish AVT receptors form a monophyletic group with the amphibian receptors (Fig. 3). Based on these results, we concluded that AB377531 and AB377532 encode the lungfish V1a-type and V2-type receptors, respectively.
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).
Therefore, we examined the effects of AVT administration on
PLC/Ca2+ mobilization and AC/cAMP synthesis in CHO cells
transfected with the cloned receptors. Addition of AVP or AVT to the cells
transfected with lungfish V1a-type receptor resulted in an increase in
intracellular Ca2+ concentration ([Ca2+]i) in
a concentration-dependent manner (Fig.
4A). On the other hand, in the CHO cells transfected with lungfish
V2-type receptor, an extremely high dose of AVP or AVT resulted in a slight
increase in [Ca2+]i; however, the responsiveness was
1000 times lower than that in the cells transfected with V1a-type receptor
(Fig. 4B). Although addition of
AVP or AVT to the cells transfected with V1a-type receptor did not increase
intracellular cAMP levels (Fig.
4C), the cells transfected with lungfish V2-type receptor
responded with the accumulation of cAMP in a concentration-dependent manner
following AVP or AVT stimulation (Fig.
4D). Addition of ghrelin as a negative control did not affect the
[Ca2+]i and cAMP levels, similar to the results in the
cells transfected with only the vector
(Fig. 4A–D).
Expression of the AVT receptors in the freshwater and estivating lungfish
To examine the effects of long-term estivation (EST) on the expression of
lungfish AVT receptors, the specimens were estivated for 90 days. In the EST
lungfish, the mean body mass decreased from 129.4±9.1 to
108.4±8.2, and the percentage change in body mass before and after the
treatment was –16.2% (Fig.
5A). The plasma osmolality and Na+ concentration in the
EST specimens was 1.5- and 1.2-fold greater, respectively, than the
corresponding values of the specimens maintained in freshwater (FW;
Fig. 5B). The plasma urea
concentration in the EST specimens increased 13.1-fold compared with the FW
specimens (Fig. 5B). Next, the
tissue expression pattern of the cloned AVT receptor mRNAs was examined by
RT-PCR in the FW and EST lungfish. In the FW lungfish, lungfish V1a-type
receptor mRNA was ubiquitously expressed in all the tissues examined and was
strongly expressed in the brain, eye, gill, lung and heart of the lungfish
(Fig. 6A). Lungfish V2-type
receptor mRNA was expressed in the brain, eye, gill, lung, heart, kidney,
testis, muscle and skin of the FW specimens and was strongly expressed in the
heart and kidney (Fig. 6A). No
amplified product for lungfish V2-type receptor mRNA was observed in the
liver, gall bladder, pancreas, intestine or ovary. In the EST lungfish, there
was no obvious change in the expression pattern and levels of V2-type receptor
mRNA between the FW and EST conditions
(Fig. 6A,B). The expression
pattern of V1a-type receptor mRNA also remained unchanged under the EST
condition; however, the expression levels of V1a-type receptor mRNA appeared
to have been increased under the EST condition in several tissues such as the
liver, gall bladder and pancreas (Fig.
6A,B).
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Immunochemical detection of lungfish V2-type AVT receptor
In the western blot analysis, a 61-kDa immunoreactive band was detected in
the membrane fractions from the heart and kidney using the affinity-purified
antibody to lungfish V2-type receptor. However, the band was not detected in
the fraction from the liver used as a negative control
(Fig. 8A). The molecular mass
of the immunoreactive band for lungfish V2-type receptor was similar to that
for the glycosylated form (47–62 kDa) of mammalian V2 receptors
(Fenton et al., 2007;
Gutkowska et al., 2007). As
shown in Fig. 8B, no
immunoreactive band was observed after preabsorption with the immunogen (1
µgm l–1). There was no significant difference between the
FW and EST lungfish with regard to the amount of V2-type receptor protein
levels (Fig. 8C), which
coincides with the result of the V2-type receptor mRNA expression.
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The renal nephron of the African lungfish P. dolloi is composed of
a glomerulus, neck segment, proximal tubule, intermediate segment, early part
of distal tubule, late part of distal tubule, and collecting tubule
(Hentschel and Elger, 1987;
Ojeda et al., 2006). In the
present study, the nephron structure of P. annectens was observed to
be similar to that of P. dolli. The kidney can be divided into three
zones (ventral, middle and dorsal). The different renal tubule segments were
identified based on the characteristics of the cell and arrangement of the
segments reported in the previous studies
(Hentschel and Elger, 1987;
Ojeda et al., 2006). The early
part of the distal tubule is mainly found in the ventral zone of the kidney,
while the dorsal zone contains the proximal tubule and the collecting tubule.
The glomerulus and the late part of the distal tubule are located in the
middle zone of the kidney (Fig.
9A,B).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Kohno et al., 2003). In the
present study, administration of AVT to CHO cells transfected with lungfish
V1a-type receptor or V2-type receptor resulted in intracellular
Ca2+ mobilization and the accumulation of cAMP in a
concentration-dependent manner, respectively. These results clearly indicate
that both AVT receptors are as functional as those in tetrapods. This is the
first direct evidence for the existence of V2-type receptor in fish,
suggesting that V2-type receptor has emerged before the appearance of
amphibians.
Balment and colleagues (Perrott et al.,
1993; Warne, 2002)
suggest that the AVT receptor associated with the AC/cAMP signaling pathway
might be present in teleosts. In renal tubules isolated from rainbow trout,
administration of AVT stimulated intracellular cAMP production in a
dose-dependent manner (Perrott et al.,
1993; Warne,
2002). However, there has been no molecular evidence for the
existence of the V2-type AVT receptor in teleosts. In the databases for the
V2-type AVT receptor sequences, we found several nucleotide sequences with
homology to known V2-type receptor sequences in the databases of puffer fish
(EMBL accession no. CAAE01014729 and CAAE01014991) and zebrafish (Genbank
accession no. XP-001346005; EMBL accession no. CAN87971). However, the
proteins deduced from these nucleotide sequences appear to be structurally
incomplete, because these nucleotide sequences represent a deletion of the
3' coding region or an elongated nucleotide sequence such as a fusion
gene. The nucleotide sequences found in a Blast search may represent
pseudogenes duplicated from an ancestral neurohypophysial hormone receptor
gene because whole-genome duplication occurred during the evolutionary process
to teleosts (Van de Peer,
2004). An exploration of the V2-type receptor in coelacanths and
elasmobranchs, which diverged earlier than lungfish, will be necessary to
elucidate the molecular evolution and physiological function of the AVP/AVT
receptor family in vertebrates.
In the present study, we found that lungfish V2-type receptor mRNA was
strongly expressed in the kidney. In mammals, the V2 receptor is predominantly
expressed in the distal convoluted tubules and collecting ducts of the kidney
and is involved in the regulation of permeability to water, Na+ and
urea in the renal tubule of the kidney
(Bentley, 2002). Kohno et al.
showed that in the Japanese tree frog, V2-type receptor mRNA was strongly
expressed in the kidney, urinary bladder and pelvic skin, where absorption of
water and ions occurs (Kohno et al.,
2003). Therefore, the high expression of lungfish V2-type receptor
in the kidney is consistent with the distribution of V2-type receptor in
tetrapods, suggesting that the V2-type receptor is important for body fluid
regulation in the kidney. The immunohistochemical study further revealed that
the V2-type receptor protein is localized in the basolateral area of the cells
of the late part of the distal tubules in the lungfish kidney. In tetrapods,
water and Na+ filtered in the glomerulus are reabsorbed in the
distal tubule and the collecting tubule, which are the target sites of AVP/AVT
(Bentley, 2002). In these
segments, the V2-type receptor mediates the accumulation of aquaporin-2 water
channel (AQP2) into the apical membrane through the activation of PKA
(Lolait et al., 1992;
Nielsen et al., 2002). In our
preliminary experiment, lungfish AQP2 (DDBJ accession no. AB474277) was
observed to be expressed on the apical cell membrane of the late part of the
distal tubules in the kidney of P. annectens (N.K., S.H. and S.
Uchiyama, unpublished results). The V2-type AVT receptor expressed in the
kidney may be functionally coupled to the water channel and may be as involved
in osmoregulation in the lungfish as it is in tetrapods.
In the lungfish, high V2-type receptor expression levels were also detected
in the heart; however, the function of V2-type receptor in this organ remains
to be elucidated. Moderate V2-type receptor expression was found in various
tissues including the brain, gill, lung and skin. Consistent with our results,
the expression of V2-type receptor mRNA has been found in various organs and
tissues, even in frogs and newts (Kohno et
al., 2003; Acharjee et al.,
2004; Hasunuma et al.,
2007). Therefore, lungfish V2-type receptor may be implicated in a
broad range of physiological functions in primitive vertebrate groups.
It has been reported that P. annectens, P. dolloi and P.
aethiopicus can experimentally estivate in mud or air for several months
(Chew et al., 2004;
Ip et al., 2005;
Loong et al., 2008). In the
present study, 90 days of EST using a plastic bag decreased the average body
mass of the lungfish by approximately 16% compared with their mass before EST.
The plasma osmolality and Na+ and urea concentrations also
increased significantly under the EST condition. It appears that water
retention is facilitated during the EST period. However, we could not detect
any obvious change in the expression levels of V1a-type and V2-type receptor
mRNA in the kidney during the 90 days of EST. Similar results in the protein
levels were found by western blot analysis with a specific antibody against
lungfish V2-type receptor. There was no obvious difference in the density of
the immunoreactive 61-kDa band in the kidney between the FW and EST lungfish.
Conversely, 90 days of EST remarkably increased the expression levels of AVT
precursor mRNA in the hypothalamus compared with that in the FW lungfish, and
the mRNA levels significantly correlated with the plasma osmolality.
Therefore, the present results suggest that the production of AVT may be
accelerated in response to an elevation in the plasma osmolality and/or
hypovolemia caused under EST. Warne et al. also reported that, although the
hypothalamic AVT precursor mRNA and plasma AVT concentrations were
significantly elevated after the hypertonic challenge of transfer of
euryhaline flounder from FW to seawater, the expression levels of the V1a-type
AVT receptor remained unchanged (Warne et
al., 2005). It has been demonstrated that the expression of the V2
receptor in the rat kidney is downregulated during dehydration
(Steiner and Phillips, 1988;
Park et al., 1998;
Machida et al., 2007), which
opposes the conservation of water and sodium in dehydration; however, the
physiological mechanisms that regulate the expression of the AVP receptors are
not clearly understood. Identification of the promoter regions and
transcriptional regulatory factors for the AVP/AVT receptors will be required
to elucidate the regulation of expression of the receptors.
In conclusion, this was the first study to detect the functional V2-type AVT receptor in African lungfish. In the EST lungfish, a significant increase in the AVT precursor mRNA level was observed in the hypothalamus. The elevation of the AVT level induced by the EST condition may contribute to an antidiuretic action through both V1a- and V2-type receptors expressed in the lungfish kidney.
LIST OF ABBREVIATIONS
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Acharjee, S., Do-Rego, J. L., Moon, J. S., Ahn, R. S., Lee, K., Bai, D. G., Vaudry, H., Kwon, H. B. and Seong, J. Y. (2004). Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors. J. Mol. Endocrinol. 33,293 -313.[Abstract]
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Gutkowska, J., Miszkurka, M., Danalache, B., Gassanov, N., Wang, D. and Jankowski, M. (2007). Functional arginine vasopressin system in early heart maturation. Am. J. Physiol. 293,H2262 -H2270.
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