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Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan
Address all correspondence and requests for reprints to: Dr. Shigeyasu Tanaka, Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan. E-mail: sbstana{at}ipc.shizuoka.ac.jp.
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Introduction |
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In this study, we identified a previously unrecognized member of the amphibian AQP family, AQP-h2, in the ventral pelvic skin of the tree frog, and demonstrated that both it and the previously identified AQP-h3 are vasotocin-regulated AQP in amphibians.
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Materials and Methods |
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Construction of the frog ventral skin cDNA library
Total RNA was prepared from 0.28 g of frog skin by using TRIZOL RNA extraction reagent (Life Technologies, Rockville, MD). Then 11.52 µg of polyadenylated [poly (A)+] RNA was selected from about 572 µg of the total RNA by using Oligotex-dT30 super (Takara, Kyoto, Japan). We constructed a 
ZAP cDNA library (9.6 x 106 plaque-forming units/µg of arms) from the poly (A)+RNA by use of a ZAP express cDNA synthesis kit and a Gigapack III Gold cloning kit (Stratagene, La Jolla, CA), in accordance with the manufacturer’s instructions.
Oligonucleotide primers for PCR
Degenerate primers for the original amplification of frog AQP fragments were designed based on the amino acid sequences around the two conserved Asn-Pro-Ala (NPA) boxes of MIP family aquaporins (20). The following primers were commercially synthesized (Life Technologies, Inc.): P1 (sense), 5'-AGCGGGG(CG)(CT)CAC(AC)T(CT)AACCC-3'; P2 (antisense), 5'-GG(AT)CC(AG)A(CT)CCA(AG)AAGA(CT)CCA-3'; P3 (antisense), 5'-A(AG)(AG)GA(CG)C(GT)(GT)GC(AT)GG(AG)TTCAT-3'.
RT-PCR amplification
The skin poly (A)+RNA (0.5 µg) was heated at 65 C for 3 min and cooled on ice. For cDNA synthesis, the denatured RNA was incubated at 42 C for 1 h in a 20 µl-reaction buffer containing Rous-associated virus 2 reverse transcriptase (9.9 U, Takara), 1 mM concentration of each deoxynucleotide triphosphate (dNTP), 7.5 mM oligo-deoxythymidine (19) primer, and ribonuclease inhibitor (20 U, Toyobo, Osaka, Japan) and then at 52 C for 30 min. Using the reverse-transcribed first-strand cDNA, we then performed PCR in 25 µl of Ex-Taq buffer containing a 0.2 mM concentration of each dNTP, 1 mM primer P1, and 2 mM primer P2 along with 0.625 U of Ex-Taq polymerase (Takara). Nested PCR amplification was further performed using primers P1 and P3. The procedure of PCR amplification consisted of an initial denaturation step of 95 C for 5 min, followed by denaturation (94 C, 90 sec), annealing (50 C, 90 sec) and extension (72 C, 150 sec) for 30 cycles in a thermal cycler (ASTEC, Fukuoka, Japan). Amplified fragments were subcloned into pGEM-3z vectors (Promega, Madison, WI), and sequenced. Because one clone with its sequence corresponding to that of Bufo AQP-t2 (AAC69694) was identified, we tentatively designated it as AQP-h2.
Screening of the frog skin cDNA library
A DNA probe, obtained from the first PCR product as described above, was synthesized by using a digoxigenin (DIG)-High Prime kit (Roche Molecular Biochemicals, Meylan, France) and used to screen the cDNA library constructed from the pelvic skin in accordance with the manufacturer’s instructions. The membrane was hybridized with DIG-labeled cDNA probes at 68 C overnight and washed twice in 1x saline sodium citrate/0.1% sodium dodecyl sulfate for 1 h at 50 C. After blocking, the membrane was incubated with alkaline phosphatase-conjugated sheep anti-DIG Fab antibody (Roche), reacted with 25 mM CSPD, disodium 3-(4-methoxyspiro{1, 2-dioxetane-3, 2'-(5'-chloro)tricyclo[3.3.1.13.7]decan}-4-yl)phenyl phosphate) chemiluminescence substrate (Tropix, Inc., PE Applied Biosystems, Foster City, CA), and then juxtaposed to on Hyperfilm-ECL (Amersham Pharmacia Biotech, Buckinghamshire, UK).
DNA sequence analysis
The nucleotide sequence was analyzed by the dideoxy chain-termination method using a Thermo Sequenase Cycle Sequencing Kit (United States Biochemical Corp., Cleveland, OH) and an Aloka DNA sequencing system [Model Lic-4200L(S), Aloka Co., Ltd., Tokyo, Japan].
RT-PCR of Hyla tissues
The tissue expression of AQP-h2 mRNA was analyzed by RT-PCR. TRIZOL reagent was used to prepare total RNA from various tree frog tissues (ventral pelvic skin, dorsal skin, urinary bladder, kidneys, brain, tongue, heart, lungs, liver, stomach, small intestine, large intestine, testes, ovaries, and blood cells). After treatment of 20 µg total RNA with deoxyribonuclease I (4 U; Takara), a 10-µg aliquot of the total RNA was reverse transcribed at 42 C for 1 h and then at 52 C for 30 min in 20 µl of reaction buffer containing a 1 mM concentration of each dNTP, 9.9 U of Rous-associated virus 2 reverse transcriptase (Takara), 20 U of ribonuclease inhibitor (Toyobo), 7.5 mM oligo-deoxythymidine (19) primer (Life Technologies, Inc.). RT-PCR was performed basically by the same method described above, by using homologous primers: P4 (sense), 5'-CTTCTGGATTGGACCTTTTG-3' (609–628 bp); and P5 (antisense), 5'-GCATTAGAGCGTAGTAATCC-3' (1027–1046 bp). The RT-PCR products were analyzed on a 2% agarose gel containing ethidium bromide (0.5 µg/ml) with Marker 6 (/Sty1 digest; Wako Pure Chemicals, Osaka, Japan) for molecular weight markers.
Southern blot analysis
To obtain cDNA probe, we performed the PCR using pBK-CMV phagemid vector containing AQP-h2 cDNA, and primers P4 and P5 for AQP-h2 as described above. DIG-labeled cDNAs were synthesized with a DIG-High Prime kit (Roche). The membrane was subsequently hybridized with DIG-labeled cDNA probes, and then hybridization signals were detected with CSPD on Hyperfilm-ECL after incubating with alkaline phosphatase-labeled anti-DIG antibody, as described above.
Osmotic water permeability of oocytes
cRNAs were prepared from linearized pBK-CMV phagemid vectors containing the entire open reading frame of AQP-h1 or AQP-h2 with XhoI (Takara) and transcribed/capped with T3 RNA polymerase (mCAP RNA Capping kit, Stratagene). Stage V and VI Xenopus oocytes were defolliculated by collagenase (1 mg/ml; Roche) and microinjected with cRNAs (5 or 50 ng) or water. After a 3-d incubation in Barth’s buffer at 18 C, the oocytes were transferred from 200 mosmol to 70 mosmol Barth’s buffer, and the osmotically elicited increase in volume was monitored at 24 C under an Olympus BX50 microscope with a x4 magnifying objective lens and a charge-coupled device camera connected to a computer. The coefficient of osmotic water permeability (Pf) was calculated from the initial slope of oocyte swelling according to the previous method (9, 22). In some experiments, HgCl2 was added to a final concentration of 0.3 mM for 10 min. In other experiments, we treated the oocytes with a mixture of cAMP (8-bromo-cAMP sodium salt; Sigma, St. Louis, MO) and 1 mM 3-isobutyl-1-methylxanthine (Sigma) for 30 min before the volume measurement. To confirm whether AQP-h2 protein was expressed in Xenopus oocytes after the injection of AQP-h2 cRNA, we evaluated AQP-h2 cRNA-injected or water-injected oocytes by Western blot analysis and immunostained as described below.
Antibody
Oligopeptide corresponding to the C-terminal amino acids 255–271 (ST-140:QEQPRRKSMELQTL) of the Hyla AQP-h2, with an amino-terminal cysteine residue, was synthesized with a Model 433A (PE Applied Biosystems, Foster City, CA). The crude peptide was purified by reverse-phase HPLC with a 0–60% linear gradient of CH3CN in 0.1% trifluoroacetic acid. Purify of the peptide was confirmed by measuring its molecular mass by mass spectrometry. The antibody was raised in a guinea pig immunized with the ST-140 peptide coupled to Keyhole limpet hemocyanin (Pierce, Rockford, IL) as described previously (23). Rabbit anti-Hyla AQP-h3 serum was characterized previously (19).
Western blot analysis
The ventral pelvic skin, back skin, kidney, and urinary bladder taken from the tree frogs were homogenized in cell lysis buffer [50 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 1% Triton X-100, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin] and centrifuged in a microcentrifuge for 5 min to remove insoluble materials. The proteins were determined with a BCA Protein Assay Kit (Pierce, Rockford, IL). The supernatant protein (10 µg) was denatured at 70 C for 10 min in denaturation buffer comprising 2% sodium dodecyl sulfate, 25 mM Tris-HCl (pH 7.5), 25% glycerol, and 0.005% bromophenol blue, subjected to electrophoresis on a 12% polyacrylamide gel; and then transferred to an Immobilon-P membrane (Millipore, Tokyo, Japan). The proteins in the membrane were reacted sequentially with anti-Hyla AQP-h2 serum diluted at 1:10,000, biotinylated antiguinea pig IgG (Jackson Immunoresearch, West Grove, PA), and streptavidin-conjugated horseradish peroxidase (DAKO Japan, Co., Ltd., Kyoto, Japan). The reaction product on the membrane was visualized by using an ECL Western blot detection kit (Amersham). As a control, the primary antibody was replaced with anti-Hyla AQP-h2 serum preincubated with 10 µg/ml of the immunogen peptide. To determine whether the immunoreactive proteins were glycosylated, we treated an extract from the urinary bladder for 24 h at 37 C with peptide-N-glycosidase F (Daiichi Pure Chemicals, Tokyo, Japan) before SDS-PAGE and Western blotting, in accordance with the manufacturer’s instructions.
Experimental protocol for stimulation with vasotocin
The ventral skin was removed from each of 13 tree frogs and bathed in frog Ringer’s solution. Under a stereomicroscope, a piece of each ventral skin was divided into four small fragments (
5 x 5 mm), and then two of the fragments were incubated with the Ringer’s solution in the presence and two in the absence of 10-8 M [Arg (8)]-vasotocin (Peptide Institute, Inc., Osaka, Japan) at 23 C for 20 min under 95% O2-5% CO2 air. After incubation, the specimens were examined by immunofluorescence microscopy.
Immunofluorescence
Several tissues including skin, kidney, and urinary bladder were quickly removed, fixed overnight in periodate-lysine-paraformaldehyde fixative, dehydrated, and embedded in Paraplast. Three-micrometer sections were cut and mounted on gelatin-coated slides. The deparaffinized sections were rinsed with distilled water and PBS. For single labeling of Hyla AQP-h2, immunofluorescence staining was performed essentially as described previously (24). The sections were sequentially incubated with 1% BSA-PBS, guinea pig anti-Hyla AQP-h2 serum (1:5000), and indocarbocyanine-labeled donkey antiguinea pig IgG (Jackson). For nuclear counterstaining, 4',6-diamidino-2-phenylindole (DAPI) was included in the secondary antibody solution. The sections were finally washed with PBS and then mounted in PermaFluor (Immunon, Pittsburgh, PA). For double-immunofluorescence staining for AQP-h2 and AQP-h3, sections were incubated with a mixture of guinea pig anti-AQP-h2 (1:5,000) and rabbit anti-AQP-h3 (1:10,000), and then reacted with a mixture of indocarbocyanine-labeled donkey antiguinea pig IgG (1:400), fluorescein isothiocyanate-labeled donkey antirabbit IgG (1:400), and DAPI. To check the specificity of the immunostaining, we performed an absorption test by preincubating the anti-AQP-h2 serum with C-terminal peptide (10 µg/ml) of AQP-h2 protein used as immunogen or with C-terminal peptide (10 µg/ml) of AQP-h3. Specimens were examined with an Olympus BX50 microscope equipped with a BX-epifluorescence attachment (Olympus Optical Co., Tokyo, Japan).
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Results |
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shows the full cDNA sequence of Hyla AQP-h2 and the deduced amino acids. The cDNA consisted of a 5'-untranslated region of 87 bp and a 3'-untranslated region of 382 bp followed by a poly (A) tail. The open reading frame encoded a protein of 268 amino acids with a relative molecular mass calculated to be 29,204 Da. Hydropathy analysis predicted six transmembrane regions with an N terminus and a C terminus localized in the cytoplasm, similar to other MIP family members (Fig. 1B). There was one putative N-linked glycosylation site at Asn-124, one protein kinase C phosphorylation site at Ser-231, and one protein kinase A phosphorylation site at Ser-262 in the AQP-h2 protein. The amino acid sequence contained the conserved NPA motifs found in all MIP family members, as well as a cysteine just upstream from the second NPA motif, which positioning is similar to that in Hyla AQP-h1 and AQP-h3 (19). Hyla AQP-h2 had its highest amino acid sequence homology to Bufo AQP-t2 (94.4%; AAC69094) and high homology to Hyla AQP-h3 (64.4%; Ref. 19), rat AQP2 (59.1%; Ref. 9), mouse AQP2 (59.1%; Ref. 25), and human (61.4%; Ref. 26) AQP2, but less homology to Hyla AQP-h1 (45.0%; Ref. 19), rat AQP3 (32.6%; Ref. 11), mouse AQP3 (32.9%; Ref. 27), and human AQP3 (31.9%; Ref. 28).
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). This RT-PCR result was confirmed by Southern blot analysis (data not shown).
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, lane 1). No band was detectable when the extracts of dorsal skin and kidney were examined (Fig. 3A, lanes 2 and 3). Furthermore, immunopositive bands were seen at 29.0 kDa and at 42.5–65.8 kDa in the case of the extract of urinary bladder (Fig. 3A, lane 4). The immunopositive bands described above were not detected when the antiserum was preabsorbed with the peptide used as the immunogen (Fig. 3B). This evidence showed that these immunoreactive bands were specific to the antiserum. To confirm that the immunoreactive bands for the urinary bladder were glycosylated, we performed a digestion experiment using peptide-N-glycosidase F. After digestion, the stained smear band became a band of 29.0 kDa, suggesting that the bands of apparent higher molecular mass represented glycosylated forms of the 29.0 kDa-Hyla AQP-h2 protein (Fig. 3C).
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, A and B). In the outermost sublayer of the stratum granulosum, AQP-h2 was localized in the basolateral membrane (Fig. 4C). In one particular experiment, immunolabels were observed in the entire plasma membrane of the granular cells of the lower granulosa sublayer. No signal was found in other types of cells including the mitochondria-rich cells and exocrine glandular cells in the pelvic skin (Fig. 4, A and B). To confirm the specificity of the staining, we carried out a control experiment. Immunopositivity for AQP-h2 in the granular cells was abolished when the antiserum was preincubated with the C-terminal peptide of Hyla AQP-h2 protein used as the immunogen (Fig. 4D). On the other hand, adsorption with the C-terminal peptide of AQP-h3 did not affect the AQP-h2 labeling (data not shown). When the urinary bladder was immunostained with the antiserum, the labeling was observed as a spot-like pattern in the cytoplasm under the apical plasma membrane of the granular cells (Fig. 4E). No labeling of the urinary bladder was obtained when the antiserum was preabsorbed with its corresponding immunogen (Fig. 4F). In addition, no positive signal for AQP-h2 was detected in kidney (Fig. 4, G and H) or in several other tissues, such as tongue, liver, stomach, small and large intestines, testis, and ovary (data not shown).
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, A and B). Swelling of AQP-h2 cRNA-injected oocytes appeared higher than that of water-injected oocytes, but no significant difference was observed between them. However, when cAMP was added to the medium containing the AQP-h2 cRNA-injected oocytes, the swelling of oocytes increased as shown in Fig. 5A. The cAMP-stimulated water permeability was completely inhibited by 0.3 mM HgCl2 (Fig. 5B). In addition, when the water-injected oocytes were stimulated with cAMP, they showed no significant increase in the water permeability. To evaluate whether AQP-h2 protein was expressed in the AQP-h2-injected oocytes, we performed a Western blot analysis on an extract of the oocytes. As shown in Fig. 5C, the AQP-h2 protein was detected as three bands at 30–32 kDa (lane 1), which were slightly higher in molecular weight than the AQP-h2 detected for the ventral skin (lane 3) or urinary bladder (lane 4). In addition, no band was detected for the extract of the water-injected oocytes (lane 2). However, when sections of the AQP-h2 cRNA-injected oocytes were immunostained with anti-Hyla AQP-h2, a large immunofluorescent mass was seen in the cytoplasm near the nucleus, but not in the plasma membranes (Fig. 5D-a). The immunopositive sites were completely eliminated by preabsorption of the antiserum with 10 µg/ml of the immunogen peptide (Fig. 5D-b). When the oocytes were stimulated by cAMP, the label for AQP-h2 was dispersed throughout the cytoplasm, but was still not seen in the plasma membrane (Fig. 5D-c). No immunolabel was found in water-injected oocytes (Fig. 5D-d).
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, A, C, and D). On the other hand, when the skin fragments were incubated in the medium with vasotocin, AQP-h2 was detected in the apical membrane in the granular cells of the outermost sublayer (Fig. 6, E, G, and H). We obtained similar results for the AQP-h3 protein (Fig. 6, B–D and F–H). In the granular cell sublayer beneath the outermost layer, the fluorescence for both AQP-h2 and AQP-h3 showed a pattern similar to that found in the nonstimulated condition, although the staining intensity was slightly weaker (Fig. 6, A, B, E, and F).
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Discussion |
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In this study, we investigated the expression of AQP-h2 mRNA by using RT-PCR. AQP-h2 was expressed in several tissues including vasotocin-dependent tissues, i.e. the ventral skin, kidney, and urinary bladder. Taken together with the data from sequence homology between AQP-h2 and mammalian AQPs, it is very likely that AQP-h2 in amphibians is the counterpart of mammalian AQP2.
Western blot analysis and digestion experiments with peptide-N-glycosidase F of the extracts of Hyla skin and bladder showed that Hyla AQP-h2 protein was present in nonglycosylated and glycosylated forms, and we clearly showed that this antibody (ST-140) was specific for Hyla AQP-h2 protein.
The immunofluorescence experiments of our study revealed that AQP-h2 was specifically expressed in the granular cells of the ventral pelvic skin and in the urinary bladder. This distribution of immunopositive sites was in good agreement with the results of the tissue distribution of mRNA found by RT-PCR. However, no immunopositive cells were found in the kidney, although AQP-h2 mRNA was detected in the kidney by RT-PCR. Thus we assumed that AQP-h2 in the Hyla kidney is scarcely translated, because AQP-h2 protein was not detected by either immunofluorescence or Western blotting analysis. However, experiments should be conducted under different physiological conditions to clarify whether AQP-h2 protein is expressed in the kidney, because expression of the protein may be detectable at a certain condition.
In response to ADH or isoproterenol, the intramembrane particles appear in the apical plasma membrane of the granular cells in the outermost granular layer of the skin, thereby increasing water permeability; intramembrane particles were therefore considered to be water channels (14, 30). It was a challenging issue to reveal molecular characterization of the intramembrane particles. In this study, we showed their molecular identity.
In the present study, we demonstrated that AQP-h2 protein was localized in the basolateral plasma membrane of granular cells in the outermost granular sublayer in situ. A similar labeling pattern was obtained after skin had been removed from the animal and incubated in the medium. In subsequent study, we investigated the immunolocalization of these AQPs in the frog ventral skin after vasotocin treatment. In the ventral skin after this treatment, signals for AQP-h2 and AQP-h3 were enhanced in the apical plasma membrane of the granular cells in the outermost granular sublayer. This sublayer is referred to as the first reacting cell layer and forms a continuous barrier between the outside and inside of the body, its continuity being preserved by tight junctions (14, 31, 32, 33). The tight junctions separate the plasma membrane into two domains, i.e. the apical membrane and basolateral membrane. In the frog skin, the tight junctions are formed as the granular cells differentiate and move upward to the surface. Accordingly, the reason why AQP-h2 or AQP-h3 was found in the entire plasma membrane of the granular cells beneath the outermost sublayer may be that tight junction formation was yet incomplete between these cells.
It is well known that, in mammals, AQP2 is involved in reabsorption of water in the collecting duct of the kidney (9). Mammalian AQP2 is expressed in the apical plasma membrane and cytoplasm just beneath the apical membrane (9, 34). In response to ADH, mammalian AQP2 is translocated from the cytoplasmic pool to the apical membrane, thereby raising the water permeability (34, 35); whereas in the nonstimulated condition, it is removed from the apical membrane by endocytosis, thereby decreasing water permeability (35). From this evidence, it has been proved that mammalian AQP2 is an ADH-regulated AQP. In the present study, we obtained evidence that AQP-h2 as well as AQP-h3 migrate to the basolateral plasma membrane in the nonstimulated condition, whereas both were translocated to the apical plasma membrane in response to vasotocin. Considering these points together, we propose that both Hyla AQP-h2 and AQP-h3 proteins are water-channel molecules that are regulated in response to vasotocin. However, the exact mechanism of sorting these AQP proteins to the apical or basolateral plasma membrane remains unclear at present.
Several lines of evidence showed that protein kinase A phosphorylation at the consensus Ser-256 is necessary for AQP2 to migrate from the intracellular membrane vesicles to the apical plasma membrane (36, 37). Because there is a protein kinase A site at Ser-262 in AQP-h2 and at Ser-255 in AQP-h3 (19), phosphorylation in these sites may be necessary for translocating AQP-h2 or AQP-h3 protein to the apical plasma membrane.
In the osmotic water permeability experiments using Xenopus oocytes, we found weak water permeability in the AQP-h2-injected oocytes. Concerning these oocytes, our Western blot analysis and immunohistochemistry revealed that AQP-h2 protein was not fully translocated to the plasma membrane of the ooctyes, thereby causing low activity of AQP-h2 in water permeability assay. A similar result was obtained for the AQP-h3-injected oocytes in a previous study (19). On the other hand, in the presence of cAMP the water permeability of AQP-h2-injected oocytes was greatly increased; and the immunoreactive AQP-2 was dispersed throughout the cytoplasm, although we were not able to detect a positive reaction in the plasma membrane. Because cAMP enhanced the water permeability in the AQP-h2-injected oocytes, a small amount of the protein, a degree that is undetectable in this immunofluorescence study, may be expressed in the plasma membrane of the oocytes. On the other hand, in the Western blot analysis using the AQP-h2-injected oocytes, we obtained immunopositive band with slightly higher molecular size than the AQP-h2 detected for the ventral skin. Consequently, such a difference in molecular sizes may be reflected on the disturbance of normal intracellular traffic for AQP-h2 protein. However, it needs to clarify the reason why sufficient amount of the AQP-h2 protein is not translocated to the plasma membrane in Xenopus oocytes. Thus, our results suggest the possibility that vasotocin-regulated membrane trafficking mechanisms or unknown factors, which exist in the Hyla ventral skin cells, but not in Xenopus oocytes, are necessary for the AQP-h2 protein to be translocated to the plasma membrane.
Taken together, our data suggest that both AQP-h2 and AQP-3 are vasotocin-regulated AQPs in amphibians, capable of being translocated from the cytoplasmic pool to the apical plasma membrane and thereby playing a role in water balance of the frog body.
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Footnotes |
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Abbreviations: ADH, Antidiuretic hormone; AQP, aquaporin; DAPI, 4',6-diamidino-2-phenylindole; DIG, digoxigenin; dNTP, deoxynucleotide triphosphate; MIP, major intrinsic protein; NPA, Asn-Pro-Ala; Pf, water permeability; poly (A)+, polyadenylated.
Received April 3, 2003.
Accepted for publication May 14, 2003.
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