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Comparative Biochemistry and Physiology, Part A 148 (2007) 72–81www.elsevier.com/locate/cbpa

Review

Amphibian aquaporins and adaptation to terrestrial environments: A review☆

Masakazu Suzuki ⁎, Takahiro Hasegawa, Yuji Ogushi, Shigeyasu Tanaka

Department of Biology, Faculty of Science, Shizuoka University, Ohya 836, Suruga ward, Shizuoka city, Shizuoka 422-8529, Japan

Received 11 May 2006; received in revised form 3 December 2006; accepted 5 December 2006Available online 16 December 2006

Abstract

In many anurans, the pelvic patch of the ventral skin and the urinary bladder are important osmoregulatory organs. Since the discovery of waterchannel protein, aquaporin (AQP), in mammalian erythrocytes, 17 distinct full sequences of AQP mRNAs have been identified in anurans.Phylogenetic tree of AQP proteins from amphibians and mammals suggested that anuran AQPs can be divided into six types: i.e. types 1, 2, 3, and5, and anuran-specific types a1 and a2. Among them, two types of anuran AQPs (types 1 and a2) are localized in the skin and urinary bladder byimmunohistochemistry. Tree frog type-a2 AQPs, AQP-h2 and AQP-h3, are vasotocin-regulated water channels predominant in the osmoregulatoryorgans. Both the AQP-h2 and AQP-h3 are expressed at the granular cells underneath the keratinized layer in the pelvic patch, whereas only AQP-h2 is detected at the granular cells in the urinary bladder. In response to vasotocin, both the molecules seem to be translocated from thecytoplasmic pool to the apical plasma membrane of the granular cells. On the other hand, type-1 AQPs, Rana FA-CHIP and Hyla AQP-h1, aredetected at the endothelial cells of blood capillaries in frog osmoregulatory organs. These findings suggest that AQP-h2 and AQP-h3 are keyplayers for transepithelial water movement, and that FA-CHIP and AQP-h1 might be important for the transport of absorbed water into the bloodflow. Comparative investigation of type-a2 AQPs in anurans further revealed that AQP-h2 and -h3-like molecules might exist at the urinarybladder and the pelvic skin, respectively, in various anurans from aquatic species to arboreal dwellers. AQP-h2-like protein is also detected in thepelvic skin of terrestrial and arboreal species. It is possible that this molecule might have occurred in the pelvic skin as anurans penetrated intodrier environments.© 2006 Elsevier Inc. All rights reserved.

Keywords: Aquaporin; Water channel; Tree frog; Pelvic patch skin; Urinary bladder; Vasotocin; Immunohistochemistry; Anurans

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732. Aquaporin family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733. Amphibian aquaporins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

3.1. Type-1 AQPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733.2. Type-a2 AQPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763.3. Transepithelial water transport through AQPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

4. Adaptation of anurans to terrestrial environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

☆ This paper was presented in the session “Water transport” at the Society of Experimental Biology's Annual Meeting at the University of Kent, Canterbury, UKApril 2nd–7th 2006.⁎ Corresponding author. Tel.: +81 54 238 4769; fax: +81 54 238 0986.E-mail address: [email protected] (M. Suzuki).

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73M. Suzuki et al. / Comparative Biochemistry and Physiology, Part A 148 (2007) 72–81

1. Introduction

Amphibians represent the first vertebrates that emerged fromaquatic habitats to terrestrial environments. To adapt to dryerenvironments, many adult anurans have evolved specializedosmoregulatory organs: the ventral pelvic patch, or seat patch, toabsorb water from the external environments and urinary bladderthat stores water and reabsorbs it in times of need (Bentley andYorio, 1979; Hillyard, 1999; Bentley, 2002). Hydromineraltransport across the tight epithelium has been studied intensive-ly, using these organs as model systems (Macknight et al., 1980;Jorgensen, 1997). Electrophysiological and pharmacologicalstudies revealed numerous key molecules such as epithelialsodium channel (ENaC) (Bentley, 1968; Garty and Palmer,1997), H+-ATPase (Harvey, 1992), cystic fibrosis transmem-brane conductance regulator (CFTR)/chloride channel (Will-umusen et al., 2002; Jensen et al., 2003), and Na+/K+-ATPase(Koefoed-Johnsen and Ussing, 1958). The CFTR in toad skinwas further identified by cDNA cloning (Amstrup et al., 2001).Considering the functions of these players and tight junction,several models are proposed to explain molecular mechanismsfor ion transport across the amphibian skin (Larsen, 1991;Jensen et al., 2003) and urinary bladder (Macknight et al., 1980).

Coupled with ion transport, water moves across the tightepithelium through two pathways, transcellular and paracellu-lar. Although the paracellular water transport through the tightjunction area occurs in amphibian epithelia (Guo et al., 2003;Orce et al., 2004), the main route is the transcellular pathway.Classically, the transcellular water transport was believed to bepassive diffusion through the lipid bilayer of cell membranes.However, biophysical, physiological, and electron microscopicstudies predicted the existence of water movement mediated bymembrane channel proteins, called aggregates (Chevalier et al.,1974; Brown et al., 1983; Yasui, 2004). During the early 1990ssuch water channel proteins were discovered, and are nowcalled aquaporins (AQP) (Agre et al., 1993). As for amphibians,approximately 20 AQP cDNAs have been registered in DNAdata bank, and it is getting more important to characterize thefunction and localization of AQPs for the understanding ofwater pathways in the amphibian body. In this review, we focuson amphibian AQPs and discuss molecular mechanisms of thetransepithelial water permeability in the pelvic patch andurinary bladder, in terms of AQPs. Furthermore, AQPs presentin the osmoregulatory organs are compared among severalanurans to consider the role of AQPs in the terrestrial adaptationof anurans. The kidney is also involved in amphibianosmoregulation (Bentley, 2002; Uchiyama and Konno, 2006),but this topic is not dealt with here.

2. Aquaporin family

Aquaporins have been discovered in a vast and varied arrayof organisms, ranging from bacteria to animals and plants.These AQPs are divided into two subfamilies: i.e. orthodoxaquaporins conducting only water and aquaglyceroporinstransporting solutes and water. Thus far, 13 isoforms of AQPs(AQP0-12) have been identified in mammals (Borgnia et al.,

1999; Takata et al., 2004; Itoh et al., 2005; Gorelick et al.,2006). AQP0 and AQP12 are unique to the lens and acinar cellsof the pancreas, respectively, whereas the other isoforms aredetected in various cells and tissues (Takata et al., 2004).Multiple isoforms of AQPs are expressed in many organs, buteach of them shows specific cellular and subcellular localiza-tion, thereby serving important physiological functions. As forthe mammalian kidney, 8 AQPs are located at specific segmentsof the nephron and other components: i.e. AQP1, AQP7,AQP8, and AQP11 at the proximal tubule (Maunsbach et al.,1997; Nejsum et al., 2000; Elkjaer et al., 2001; Morishita et al.,2005), AQP1 at the descending thin limb of Henle's loop(Maunsbach et al., 1997), AQP2, AQP3, AQP4, AQP6, andAQP8 at the collecting duct (Ishibashi et al., 1994; Frigeri et al.,1995; Nielsen et al., 1995a; Ohshiro et al., 2001; Elkjaer et al.,2001), AQP3 at the renal pelvis (Matsuzaki et al., 1999), andAQP1 at the vasa recta (Sabolic et al., 1992). Among theseAQPs, AQP2 is the vasopressin-sensitive water channel thatfacilitates water reabsorption in response to antidiuretichormone, vasopressin, by translocating from intracellularvesicles to the apical membrane of collecting duct principalcells (Nielsen et al., 1995a; Noda and Sasaki, 2005; Valentiet al., 2005). AQP2 mutations and disruption of AQP2 genecause nephrogenic diabetes insipidus, a disease characterized bya massive loss of water through the kidney (Deen et al., 1994;Yang et al., 2001). Other renal AQPs are also shown to playcritical roles in water reabsorption and urine concentration inthe kidney (Verkman, 2006).

3. Amphibian aquaporins

The full sequences of 17 AQP cDNAs have been elucidatedin anurans, but only expressed sequence tags (ESTs) areregistered in urodeles. A phylogenetic analysis suggested thatanuran AQPs can be assigned to six clusters: types 1, 2, 3, and 5,and two anuran-specific types, designated as a1 and a2 (Theletter “a” represents anuran) (Fig. 1). The cluster of type-a1AQPs is composed of AQPxlo from Xenopus laevis oocytes(Virkki et al., 2002) and another X. laevis AQP (accessionnumber: BC090201). The cluster of type-a2 AQPs containsAQP-h2 (Hasegawa et al., 2003) and AQP-h3 from the frog,Hyla japonica (Tanii et al., 2002), and AQP-t2 (AF020621)and AQP-t3 from the toad, Bufo marinus (AF020622). As foranuran osmoregulatory organs, little information is available forthe types 2, 3, 5, and a1. Therefore, this section summarizespublished data on the type-1 AQPs and type-a2 AQPs,especially highlighting the structure and function of HylaAQPs.

3.1. Type-1 AQPs

The AQP1 cluster includes mouse AQP1 (Moon et al.,1995), AQP-h1 from H. japonica (Hasegawa et al., 2003), FA-CHIP from Rana esculenta (Abrami et al., 1994), and AQP-t1from B. marinus (Ma et al., 1996) (Fig. 1). Hyla AQP-h1 iscomposed of 271 amino acid residues, and its predictedstructure is shown in Fig. 2A. Hydropathy analysis has

Fig. 1. Phylogenetic tree of AQP proteins from amphibians and mammals. Anuran AQPs are classified into six types, two of which (types a1 and a2) are unique to theanurans. A neighbour joining tree was generated using Clustal W (Thomopson et al., 1994), and transformed so that the branching pattern and clustering becomeevident. The length of each branch is not proportional to the estimated number of amino acid substitutions. The organism and the name are indicated for each AQP. Theaccession number is also shown for each AQP, except for Hyla AQP-h2K that has been recently identified in our laboratory.

74 M. Suzuki et al. / Comparative Biochemistry and Physiology, Part A 148 (2007) 72–81

predicted the presence of six transmembrane regions, as in otheraquaporins. The sequence of arginine, proline, and alanine iscalled NPA motif, and AQP-h1 contains two NPA motifs. Aseries of studies on the atomic structure of rat AQP1 revealed aselective mechanism for water permeation through a channelpore (Yasui, 2004; de Groot and Grubmuller, 2005). As for ratAQP1, a pair of NPA motifs is located in the centre of thepore, and forms a specific passage for water molecules. TheseNPA motifs and other amino acid residues important for poreselectivity of mammalian AQP1, i.e. Phe, His, Cys, and Arg(de Groot and Grubmuller, 2005), are conserved in Hyla AQP-h1 (Fig. 2A). Therefore, AQP-h1 seems to conserve the basicarchitecture for the water channel.

Physiological properties of AQP-h1 were assessed byexpressing it in Xenopus oocytes (Hasegawa et al., 2003). Theoocytes were injected with AQP-h1 cRNA, and transferred tohypoosmotic (70 mOsm) Barth's solution, and then the oocytesstarted to swell. The swelling was significantly higher than that

of water-injected oocytes, and the coefficient of osmotic waterpermeability, Pf, of AQP-h1 was about 18 times greater than thecontrol (Hasegawa et al., 2003). In addition, the enhanced waterpermeability was inhibited by 0.3 mM HgCl2 by 48%. Theseresults confirm that AQP-h1 functions as a water channel.

AQP-h1 mRNAwas detected not only in the pelvic skin, butalso in other various tissues, except blood cells and liver(Hasegawa et al., 2003). By immunohistochemistry, rat AQP1 isshown to exist at continuous capillaries in diverse tissues(Nielsen et al., 1993), and its immunopositive labels aredetected on both the apical and basolateral membranes ofnonfenestrated endothelium of descending vasa recta in thekidney (Nielsen et al., 1995b). In human kidney, AQP1 residesat the endothelium of fenestrated peritubular capillaries, as wellas fenestrated glomerular capillaries (Maunsbach et al., 1997).As for the amphibian osmoregulatory organs, AQP-h1 wasobserved along the blood vessels in the skin and urinarybladder, and along the mesothelium in the urinary bladder

Fig. 2. The deduced amino acid sequences and putative membrane topologies of AQP-h1 (A), AQP-h2 (B), and AQP-h3 (C) with six membrane-spanning alpha-helices (I–VI). The red overlay highlights NPA motifs, and the phenylalanine, histidine, cysteine, and arginine residues in thick circles are putative amino acidsimportant for aromatic/arginine constriction in the water-specific channels. The arrow, diamond, star, and triangle indicate N-glycosylation sites, mercurial inhibitionsites, and phosphorylation sites for protein kinase A and protein kinase C, respectively.


Fig. 4. Double-immunofluorescence staining against AQP-h2 (A) and AQP-h3(B) in a wax section (4 μm) of the ventral pelvic skin of tree frog. Nomarskidifferential interference-contrast image (C) is shown as the correspondingreference. Strong labels for AQP-h2 with Cy3 (red) and for AQP-h3 with FITC(green) are colocalized along the contour of granular cells of the stratumgranulosum. Nuclei are counterstained with DAPI (blue). Asterisk: blood cell.The scale bar=10 μm.

Fig. 3. Indirect immunofluorescence staining against AQP-h1 in wax sections (4 μm) of the ventral pelvic skin (A) and urinary bladder (B) of tree frog.Immunoreactivity for AQP-h1 with Cy3 (red) appears along the blood vessels (arrows) and mesothelium (arrowheads). Cell nuclear DNA is stained with DAPI (blue).The scale bar=10 μm.


(Fig. 3), like another frog aquaporin, FA-CHIP, fromR. esculenta(Abrami et al., 1997). Although morphology of the subepithelialcapillaries in amphibian pelvic skin and urinary bladder has notbeen documented in detail, physiological studies indicated thathigh rates of water uptake across the pelvic patch are maintainedwith a functional circulation (Parsons et al., 1993), and thatwater, osmotically absorbed through the pelvic patch, enterssubcutaneous blood capillaries rather than lymphatic system(Word and Hillman, 2005). Therefore, it is possible that AQP-h1,as well as FA-CHIP, plays a role in the transport of absorbedwater into the blood stream in the pelvic patch and urinarybladder. These molecules may also mediate water movementinto the body through the bladder mesothelium. It is furtherreported that gene expression of frog FA-CHIP was enhanced inthe skin and urinary bladder in response to salt acclimation(Abrami et al., 1995), although expression of toad AQP-t1mRNA was not changed by a 3-day dehydration (Ma et al.,1996).

3.2. Type-a2 AQPs

The AQPa2 cluster contains AQP-h2 (Hasegawa et al., 2003)and AQP-h3 from H. japonica (Tanii et al., 2002), and AQP-t2(AF020621) and AQP-t3 from B. marinus (AF020622) (Fig. 1).Hyla AQP-h2 consists of 268 amino acid residues (Hasegawaet al., 2003), and its putative topology is shown in Fig. 2B. Apair of canonical NPA motifs is conserved, and other aminoacids important for the selective filter for water, e.g. Phe-56,His-180, Cys-189, and Arg-195 (de Groot and Grubmuller,2005), are also conserved (Fig. 2B). In addition, one possible N-linked glycosylation site at Asn-124, one protein kinase C phos-phorylation site at Ser-231, and one protein kinase A phosphor-ylation site at Ser-262 are predicted (Fig. 2B). Likewise, HylaAQP-h3 consists of 271 amino acid residues, and has similarfeatures to Hyla AQP-h2, such as NPA motifs, glycosylationsites, and phosphorylation sites (Tanii et al., 2002) (Fig. 2C). Asmammalian AQP2 is nearly confined to the kidney (Takataet al., 2004), AQP-h2 and AQP-h3 genes are expressedabundantly in the osmoregulatory organs of the tree frog: bothAQP-h2 and-h3 mRNAs are expressed in the pelvic patch skin,

whereas AQP-h2 gene alone is expressed in the urinary bladder(Tanii et al., 2002; Hasegawa et al., 2003).

In Western blot analysis, the anti-AQP-h2 antibody detecteda major band at 29.0 kDa in both the pelvic patch and urinarybladder (Hasegawa et al., 2003). Signals were further seen at42.5–65.8 kDa in the urinary bladder. Because these signalsbecame weak after the treatment with peptide-N-glycosidase,this smear band seems to represent glycosylated forms of thismolecule, like those of Rana FA-CHIP (Abrami et al., 1997).As for the anti-AQP-h3 antibody, a major band at 29.0 kDa and

Fig. 5. Indirect immunofluorescence staining against AQP-h2 in wax sections (3 μm) of the urinary bladder (A) of tree frog. Nomarski differential interference-contrastimage (B) is shown as the corresponding reference. Immunoreactivity for AQP-h2 with Cy3 (red) appears in the subapical region of granular cells. Nuclei arecounterstained with DAPI (blue). L: lumen. Asterisk: blood cell. The scale bar=10 μm.


its glycosylated forms were detected in the pelvic patch, but noband was detectable in the urinary bladder (Tanii et al., 2002).

Immunostaining of the pelvic patch and urinary bladderwas carried out using the AQP-h2 and AQP-h3 antibodies.Before showing the results, we explain the morphology of thepelvic patch skin. The epidermis consists of four successivelayers: the stratum corneum, stratum granulosum, stratumspinosum, and stratum germinativum (Larsen, 1991). Tightjunctions exist between keratinized cells and also between theoutermost granular cells (Farquhar and Palade, 1965). Ingeneral, the stratum corneum is highly permeable to waterbecause it is composed of dying keratinized cells. Therefore,

Fig. 6. Immunofluorescence localization of AQP-h2 (A) and AQP-h3 (B) in theabdominal skin of tree frog, after AVT stimulation. The pelvic skin had beenincubated with AVT (10−8 M) at 23 °C for 20 min before a wax section (4 μm) ofthe pelvic skin was double-immunostained for AQP-h2 with Cy3 (red) and forAQP-h3 with FITC (green). Strong labels for both AQP-h2 and AQP-h3appeared along the apical membrane of first reacting granular cells of the stratumgranulosum. Nomarski differential interference-contrast image (C) is shown asthe corresponding reference. Nuclei are counterstained with DAPI (blue). Thescale bar=10 μm.

it is the more inner granular cell layer, called first-reacting celllayer, which plays a key role in controlling water transport(Voute and Ussing, 1968). Mitochondria-rich cells are presentin the stratum granulosum. By immunofluorescence staining,labels for both AQP-h2 and AQP-h3 were localized in two orthree layers of granular cells in the stratum granulosum(Hasegawa et al., 2003) (Fig. 4). These immunolabels werestrongest along the contours of granular cells, and AQP-h2appeared to be colocalized with AQP-h3 (Hasegawa et al.,2003) (Fig. 4). No signal was found in the mitochondria-richcells.

As for the urinary bladder, its epithelium is a transitional type,and two types of cells are observed on the luminal surface: viz.granular cells and mitochondria-rich cells (Macknight et al.,1980). Tubular and spherical vesicles are present in thesubapical region of the granular cell (Macknight et al., 1980;Hasegawa et al., 2005). When the urinary bladder wasimmunostained with the anti-AQP-h2 antibody, the labelingshowed a spot-like pattern in the cytoplasm under the apicalplasma membrane of the granular cells (Hasegawa et al., 2005)

Fig. 7. Immunofluorescence localization of phosphorylated AQP-h2 in theabdominal skin of tree frog before (A) and after AVT stimulation (B). The pelvicskin had been incubated with AVT (10−8 M) at 23 °C for 20 min before a waxsection (4 μm) of the pelvic skin was immunostained for phosphorylated AQP-h2with Cy3 (red). Immunopositive labels appeared along the apical plasmamembrane of first reacting granular cells of the stratum granulosum only afterAVT stimulation. Nuclei are counterstained with DAPI (blue). The scalebar=10 μm.

Fig. 8. Time course of the osmotic swelling of Xenopus oocytes microinjectedwith AQP-h3 cRNA. Before the volume measurement, some of the AQP-injected oocytes were incubated with no additive, some with 0.3 mM HgCl2,some with 8-bromo-cAMP alone, and some with 8-bromo-cAMP plus 0.3 mMHgCl2. Oocytes were microinjected also with water. Six to seven oocytes wereused in each experimental group.


(Fig. 5). By immuno-electron microscopy, AQP-h2 protein wasfurther localized on the tubular or spherical vesicles in thegranular cells (Hasegawa et al., 2005). The labels were rarelyobserved on the plasma membrane. On the other hand, AQP-h3antigens were not detected in the urinary bladder, which isconsistent with the results by RT-PCR andWestern blot analysis.

To examine whether the cellular distribution of AQP-h2and AQP-h3 is regulated by vasotocin, further study has beencarried out in vitro, using the isolated ventral skin and urinarybladder. When the skin fragments were incubated withvasotocin for 15–20 min, prominent labels for both AQP-h2and AQP-h3 occurred along the apical membrane of the firstreacting granular cells (Hasegawa et al., 2003) (Fig. 6). In thesublayers of granular cells, the signal distribution appearedsimilar to that in the control (Hasegawa et al., 2003) (Figs. 4and 6), although the fluorescence intensity was slightlyweaker. In the urinary bladder, the difference caused byvasotocin was less dramatic; spotted immunofluorescentlabeling in the apical cytoplasm of the granular cells did notseem to change, but dotted labels appeared on the apicalplasma membrane after the stimulation with AVT (Hasegawaet al., 2005). This translocation of AQP-h2 was corroboratedby immunogold electron microscopy (Hasegawa et al., 2005).After AVT stimulation, immunopositive gold particles werelocalized to a part of the luminal membrane in addition to theintracellular vesicles.

To investigate whether phosphorylation is involved in thetranslocation of frog AQP, the behavior of phosphorylated AQP(pAQP)-h2 protein in the urinary bladder has been analyzedbefore and after AVT stimulation, using a specific antibodyagainst the phosphorylated C-terminal of AQP-h2 (Hasegawaet al., 2005). By immunoblot analysis, pAQP-h2 was barelydetected before stimulation. After AVT treatment, two bands,glycosylated form (51.7–60.6 kDa) and non-glycosylated form(28.3 kDa) were detected. The amount of pAQP-h2 increasedrapidly, reaching its maximal level within 2 min. Immuno-electron microscopy detected scanty labels for pAQP-h2 on asmall number of vesicles in the granular cells when the urinarybladder was not stimulated with AVT (Hasegawa et al., 2005).After AVT stimulation, however, immunopositive labels wereobserved at the apical membrane of the granular cells, as well ason tubular or spherical vesicles. On the apical membrane, thelabels appeared to form clusters (Hasegawa et al., 2005),presumably corresponding to the location of aggregatesreported by freeze-fracture electron microscopy (Chevalieret al., 1974; Kachadorian et al., 1990). Labeling density forpAQP-h2 also changed in response to AVT, and increasedat both the intracellular compartments and the apical plas-ma membrane. As for the pelvic patch skin, strong labelsfor pAQP-h2 occurred along the apical plasma membraneof the first reacting granular cells after AVT stimulation(Fig. 7).

Transmembrane water flow through AQP-h2 and AQP-h3was evaluated by expressing their cRNAs in Xenopus oocytes(Tanii et al., 2002; Hasegawa et al., 2003). As for AQP-h2, theswelling appeared higher than that of water-injected oocytes,but no significant difference was observed between them.

However, when 8-bromo-cAMP was added to the medium, theswelling of oocytes increased very much (Hasegawa et al.,2003). The Pf of AQP-h2 was about three times greater than thecontrol. Similar results were obtained for AQP-h3 (Fig. 8).These findings suggest that both AQP-h2 and AQP-h3 are waterchannels, and that translocation of these AQPs may be regulatedby cAMP, and protein kinase A (PKA) (Kachadorian et al.,1987; Valenti et al., 2005).

3.3. Transepithelial water transport through AQPs

Based on the reports on anuran AQPs and mammalian AQP2(Kachadorian et al., 1987; Kohno et al., 2003; Noda and Sasaki,2005), we consider that frog AQPs are involved in transcellularwater transport across the tight epithelia in the following fashion(Fig. 9). In the urinary bladder (Fig. 9, right), the majority ofAQP-h2 is stored on intracellular vesicles under the apicalmembrane of the granular cells. In response to AVT, PKA isactivated and phosphorylates AQP-h2, which induces thetranslocation of AQP-h2-containing vesicles towards the apicalmembrane, and enhances the water permeability. In the pelvicpatch (Fig. 9, left), both AQP-h2 and AQP-h3 are present, andappeared located along the whole plasma membrane of thegranular cells during normal hydration. Presumably, as in thebladder, they are also contained in the intracellular vesicles.Again as in the bladder, AVT activates PKA in granular cells,which phosphorylates AQP-h2 and probably AQP-h3 as well.Intracellular vesicles containing phosphorylated AQPs thenbecome translocated onto the apical membrane, increasing thewater permeability. The absorbed water moves into the bloodcapillaries through AQP-h1, and enters the systemic circulation,leading to rehydration of the whole body.

4. Adaptation of anurans to terrestrial environments

The effects of antidiuretic hormone on osmotic watertransfer across anuran pelvic skin had been extensively

Fig. 9. Model for translocation of Hyla AQP-h2 and AQP-h3 by AVT in the first reacting granular cells of the ventral pelvic skin (left) and in the granular cells of theurinary bladder (right). AVP increases water permeability by binding to V2-type AVT receptors (Kohno et al., 2003) located on the basolateral plasma membrane ofgranular cells. The ligand-occupied AVT receptors activate adenylyl cyclase (AC), increasing intracellular cAMP levels, which leads to activation of protein kinase A(PKA). The PKA then phosphorylates AQP-h2 in the ventral pelvic skin, and AQP-h2 and/or AQP-h3 in the urinary bladder, promoting translocation of the AQP-bearing vesicles onto the apical membrane. Water flow is represented by blue arrows. AQP-X is a putative aquaporin located at the basolateral membrane of thegranular cells of the urinary bladder. Tight junctions (TJs) exist between the outermost granular cells. Although the subcellular localization of AQP-h1 is not clear,AQP-h1 might be located on the whole plasma membrane of the endothelial cells of capillaries, like mammalian AQP1, and mediate the transport of absorbed waterinto the blood stream. It is unknown whether the subepidermal capillaries are fenestrated or unfenestrated.


investigated in vitro (Warburg, 1995; Bentley, 2002). Inter-estingly, the response to antidiuretic hormone tends to begreater in the anuran species normally occupying drier habitatsthan those from wet habitats (Bentley, 1974, 2002). Toexamine the involvement of AQPs in this difference,immunoblot analysis has been performed for the tissues offive Japanese anurans. According to their habitats, they can bedivided into four groups (Table 1). X. laevis is aquatic, andRana catesbeiana and Rana nigromaculata are classified

Table 1Phylogenetic distribution of AQP-h2-like protein and AQP-h3-like mRNA in the ab

Species Habitat AQP

Xenopus laevis AquaticRana nigromaculata SemiterrestrialRana catesbeiana SemiterrestrialBufo japonicus TerrestrialHyla japonica Arboreal

into a semiterrestrial group. Bufo japonicus and H. japonicaexpand their habitats to the drier land, and are classified as aterrestrial species and tree-adapted species, respectively.Because the antibody against Hyla AQP-h2 was raised andutilized in the previous experiments (Hasegawa et al., 2003),this antibody was applied to Western blot analysis for otheranurans. AQP-h2 or AQP-h2-like protein was detected in theurinary bladder of all the species examined (Table 1).Surprisingly, the AQP-h2 homologue was detected in the

dominal skin and urinary bladder of anurans

Urinary bladder Pelvic skin

h2-like protein h2-like protein h3-like mRNA

+ − ++ − ++ − ++ + ++ + +


pelvic skin of the terrestrial anuran, as in the tree-adaptedanuran, but not in the other species (Table 1). On the otherhand, AQP-h3-like cDNAwas identified in the ventral skin ofall the anurans examined, by molecular cloning (Table 1).Therefore, AQP-h2 seems to be a urinary bladder-type AQP,while AQP-h3 seems to be a ventral skin-type AQP. As pre-viously mentioned, AQP-h2-like protein was detected also inthe pelvic skin of the terrestrial and arboreal anurans, but not inmore aquatic species. It is possible that as anurans penetratedinto drier terrestrial environments, the urinary bladder-typeAQP might have occurred in the pelvic skin, and begun totransport water together with AQP-h3.

Acknowledgements

This work was supported by Grants-in Aid from the Ministryof Education, Science, Sports, and Culture of Japan, to MS andST.

References

Abrami, L., Simon, M., Rousselet, G., Berthonaud, V., Buhler, J.M., Ripoche, P.,1994. Sequence and functional expression of an amphibian water channel,FA-CHIP: a new member of the MIP family. Biochim. Biophys. Acta 1192,147–151.

Abrami, L., Capurro, C., Ibarra, C., Parisi, M., Buhler, J.-M., Ripoche, P., 1995.Distribution of mRNA encoding the FA-CHIP water channel in amphibiantissues: effects of salt adaptation. J. Membr. Biol. 143, 199–205.

Abrami, L., Gobin, R., Berthonaud, V., Thanh, H.L., Chevalier, J., Ripoche, P.,Verbavatz, J.M., 1997. Localization of the FA-CHIP water channel in frogurinary bladder. Eur. J. Cell Biol. 73, 215–221.

Agre, P., Preston, G.M., Smith, B.L., Jung, J.S., Raina, S., Moon, C., Guggino,W.B., Nielsen, S., 1993. Aquaporin CHIP: the archetypal molecular waterchannel. Am. J. Physiol. 265, F463–F476.

Amstrup, J., Froslev, J., Willumusen, J.N., Mobjerg, N., Jespersen, A., Larsen,E.H., 2001. Expression of cystic fibrosis transmembrane conductanceregulator in the skin of the toad, Bufo bufo and possible role for Cl−

transport across the heterocellular epithelium. Comp. Biochem. Physiol. A130, 539–550.

Bentley, P.J., 1968. Amiloride: a potent inhibitor of sodium transport across thetoad bladder. J. Physiol. Lond. 195, 317–333.

Bentley, P.J., 1974. Actions of neurohypophysial peptide in amphibians, replies,and birds. In: Geiger, S.R. (Ed.), Handbook of Physiology. EndocrinologyIV, part 1. Am. Physiol. Soc., Washington, pp. 545–563.

Bentley, P.J., Yorio, T., 1979. Do frogs drink? J. Exp. Biol. 79, 41–46.Bentley, P.J., 2002. The amphibia. In: Bentley, P.J. (Ed.), Endocrines and

Osmoregulation. A Comparative Account in Vertebrates, vol. 39. Springer,Berlin, pp. 155–186.

Borgnia, M., Nielsen, S., Engel, A., Agre, P., 1999. Cellular and molecularbiology of the aquaporin water channels. Annu. Rev. Biochem. 68, 425–458.

Brown, D., Grosso, A., DeSousa, R.C., 1983. Correlation between water flowand intramembrane particle aggregates in toad epidermis. Am. J. Physiol.245, C334–C342.

Chevalier, J., Bourguet, J., Hugon, J.S., 1974. Membrane associated particles:distribution in frog urinary bladder epithelium at rest and after oxytocintreatment. Cell Tissue Res. 152, 129–140.

Deen, P.M., Verdijk, M.A., Wieringa, B., Monnens, L.A., van Os, C.H., vanOost, B.A., 1994. Requirement of human renal water channel aquaporin-2for vasopressin-dependent concentration of urine. Science 264, 92–95.

de Groot, B.L., Grubmuller, H., 2005. The dynamics and energetics of waterpermeation and proton exclusion in aquaporins. Curr. Opin. Struck. Biol. 5,176–183.

Elkjaer, M.L., Nejsum, L.N., Gresz, V., Kwon, T.H., Jensen, U.B., Frokiaer, J.,Nielsen, S., 2001. Immunolocalization of aquaporin-8 in rat kidney,

gastrointestinal tract, testis, and airways. Am. J. Physiol. Renal. FluidElectrolyte Physiol. 281, F1047–F1057.

Farquhar, M.G., Palade, G.E., 1965. Cell junctions in amphibian skin. J. CellBiol. 26, 263–291.

Frigeri, A., Gropper, M.A., Turck, C.W., Verkman, A.S., 1995. Immunoloca-lization of the mercurial-insensitive water channel and glycerol intrinsicprotein in epithelial cell plasma membranes. Proc. Natl. Acad. Sci. U. S. A.92, 4328–4331.

Garty, H., Palmer, L.G., 1997. Epithelial sodium channels: function, structure,and regulation. Physiol. Rev. 77, 359–396.

Guo, P., Hillyard, S.D., Fu, B.M., 2003. A two-barrier compartment model forvolume flow across amphibian skin. Am. J. Physiol., Regul. Integr. Comp.Physiol. 285, R1384–R1394.

Gorelick, D.A., Praetorius, J., Tsunenari, T., Nielsen, S., Agre, P., 2006.Aquaporin-11: a channel protein lacking apparent transport functionexpressed in brain. BMC Biochemistry 7, 14.

Harvey, B.J., 1992. Energization of sodium absorption by the H+-ATPase pumpin mitochondria-rich cells of frog skin. J. Exp. Biol. 172, 289–309.

Hasegawa, T., Tanii, H., Suzuki, M., Tanaka, S., 2003. Regulation of waterabsorption in the frog skins by two vasotocin-dependent water-channelaquaporins, AQP-h2 and AQP-h3. Endocrinology 144, 4087–4096.

Hasegawa, T., Suzuki, M., Tanaka, S., 2005. Immunocytochemical studies ontranslocation of phosphorylated aquaporin-h2 protein in granular cells of thefrog urinary bladder before and after stimulation with vasotocin. Cell TissueRes.. 322, 407–415.

Hillyard, S.D., 1999. Behavioral, molecular, and integrative mechanisms ofamphibian osmoregulation. J. Exp. Zool. 283, 662–674.

Ishibashi, K., Sasaki, S., Fushimi, K., Uchida, S., Kuwahara, M., Saito, H.,Furukawa, T., Nakajima, K., Yamaguchi, Y., Gojobori, T., 1994. Molecularcloning and expression of a member of the aquaporin family withpermeability to glycerol and urea in addition to water expressed at thebasolateral membrane of kidney collecting duct cells. Proc. Natl. Acad. Sci.U. S. A. 91, 6269–6273.

Itoh, T., Rai, T., Kuwahara, M., Ko, S.B., Uchida, S., Sasaki, S., Ishibashi, K.,2005. Identification of a novel aquaporin, AQP12, expressed in pancreaticacinar cells. Biochem. Biophys. Res. Commun. 330, 832–838.

Jensen, L.J., Willumusen, N.J., Amstrup, J., Larsen, E.H., 2003. Proton pump-driven cutaneous chloride uptake in anuran amphibia. Biochim. Biophys.Acta 1618, 120–132.

Jorgensen, C.V., 1997. 200 years of amphibian water economy: from RobertTowson to the present. Biol. Rev. Camb. Philos. Soc. 72, 153–237.

Kachadorian, W.A., Coleman, R.A., Wade, J.B., 1987. Water permeability andparticle aggregates in ADH-, cAMP-, and forskolin-treated toad bladder.Am. J. Physiol. 253, F120–F125.

Kachadorian, W.A., Spring, K.R., Shinowara, N.L., Muller, J., Palaia, T.A.,DiScala, V.A., 1990. Effects of serosal hypertonicity on water permeabilityin toad urinary bladder. Am. J. Physiol. 258, C871–C878.

Koefoed-Johnsen, V., Ussing, H., 1958. The nature of the frog skin potential.Acta Physiol. Scand. 42, 298–308.

Kohno, S., Kamishima, Y., Iguchi, T., 2003. Molecular cloning of an anuranV(2) type [Arg(8)] vasotocin receptor and mesotocin receptor: functionalcharacterization and tissue expression in the Japanese tree frog (Hylajaponica). Gen. Comp. Endocrinol. 132, 485–498.

Larsen, E.H., 1991. Chloride transport by high-resistance heterocellularepithelia. Physiol. Rev. 71, 235–283.

Ma, T., Yang, B., Verkman,A.S., 1996. cDNAcloning of a functional water channelfrom toad urinary bladder epithelium. Am. J. Physiol. 271, C1699–C1704.

Macknight, A.D., DiBona, D.R., Leaf, A., 1980. Sodium transport across toadurinary bladder: a model “tight” epithelium. Physiol. Rev. 60, 615–715.

Matsuzaki, T., Suzuki, T., Koyama, H., Tanaka, S., Takata, K., 1999. Waterchannel protein AQP3 is present in epithelia exposed to the environment ofpossible water loss. J. Histochem. Cytochem. 47, 1275–1286.

Maunsbach, A.B., Marples, D., Chin, E., Ning, G., Bondy, C., Agre, P., Nielsen,S., 1997. Aquaporin-1 water channel expression in human kidney. J. Am.Soc. Nephrol. 8, 1–14.

Moon, C., Williams, J.B., Preston, G.M., Cipeland, N.G., Gilbert, D.J., Nathans,D., Jenkins, N.A., Agre, P., 1995. The mouse aquaporin-1. Genomics 30,354–357.


Morishita, Y., Matsuzaki, T., Hara-chikuma, M., Andoo, A., Shimono, M.,Matsuki, A., Kobayashi, K., Ikeda, M., Yamamoto, T., Verkman, A.,Kusano, E., Ookawara, S., Takata, K., Sasaki, S., Ishibashi, K., 2005.Disruption of aquaporin-11 produces polycystic kidneys following vacuo-lization of the proximal tubule. Mol. Cell. Biol. 25, 7770–7779.

Nejsum, L.N., Elkjaer, M., Hager, H., Frokiaer, J., Kwon, T.H., Nielsen, S.,2000. Localization of aquaporin-7 in rat and mouse kidney using RT-PCR,immunoblotting, and immunocytochemistry. Biochem. Biophys. Res.Commun. 277, 164–170.

Nielsen, S., Smith, B.L., Christensen, E.I., Agre, P., 1993. Distribution of theaquaporin CHIP in secretary and resorptive epithelia and capillaryendothelia. Proc. Natl. Acad. Sci. U. S. A. 90, 7275–7279.

Nielsen, S., Chou, C.L., Marples, D., Christensen, E.I., Kishore, B.K., Knepper,M.A., 1995a. Vasopressin increases water permeability of kidney collectingduct by inducing translocation of aquaporin-CD water channels to plasmamembrane. Proc. Natl. Acad. Sci. U. S. A. 92, 1013–1017.

Nielsen, S., Pallone, T., Smith, B.L., Christensen, E.I., Agre, P., Maunsbach,A.B., 1995b. Aquaporin-1 water channels in short and long loop descendingthin limbs and in descending vasa recta in rat kidney. Am. J. Physiol. 268,F1023–F1037.

Noda, Y., Sasaki, S., 2005. Trafficking mechanism of water channel aquaporin-2. Biol. Cell 97, 885–892.

Ohshiro, K., Yaoita, E., Yoshida, Y., Fujinaka, H., Matsuki, A., Kamiie, J.,Kovalenko, P., Yamamoto, T., 2001. Expression and immunolocalization ofAQP6 in intercalated cells of the rat kidney collecting duct. Arch. Histol.Cytol. 64, 329–338.

Orce, G., Castillo, G., Chanampa, Y., Bellomio, A., 2004. Permeability towater in a tight epithelium: possible modulating action of gap junctions.Can. J. Physiol. Pharm. 82, 417–421.

Parsons, R.H., McDevitt, V., Aggerwal, V., Le Blang, T., Manley, K., Kim, N.,Lopez, J., Kenedy, A.A., 1993. Regulation of pelvic patch water flow inBufo marinus: role of bladder volume and ANG II. Am. J. Physiol. 264,R1260–R1265.

Sabolic, I., Valenti, G., Verbavatz, J.M., Van Hoek, A.N., Verkman, A.S.,Ausiello, D.A., Brown, D., 1992. Localization of the CHIP28 water channelin rat kidney. Am. J. Physiol. 263, 1225–1233.

Takata, K., Matsuzaki, T., Tajika, Y., 2004. Aquaporins: water channel proteinsof the cell membrane. Prog. Histochem. Cytochem. 39, 1–83.

Tanii, H., Hasegawa, T., Hirakawa, N., Suzuki, M., Tanaka, S., 2002. Molecularand cellular characterization of a water-channel protein, AQP-h3, specifi-cally expressed in the frog ventral skin. J. Membr. Biol. 188, 43–53.

Thomopson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTALW. NucleicAcids Res. 22, 4673–4680.

Uchiyama, M., Konno, N., 2006. Hormonal regulation of ion and watertransport in anuran amphibians. Gen. Comp. Endocrinol. 147, 54–61.

Valenti, G., Procino, G., Tamma, G., Carmosino, M., Svelto, M., 2005.Minireview: aquaporin 2 trafficking. Endocrinology 146, 5063–5070.

Verkman, A.S., 2006. Roles of aquaporins in kidney revealed by transgenicmice. Semin. Nephrol. 26, 200–208.

Virkki, L.V., Franke, C., Somieski, P., Boron, W.F., 2002. Cloning andfunctional characterization of a novel aquaporin from Xenopus laevisoocytes. J. Biol. Chem. 277, 40610–40616.

Voute, C.L., Ussing, H.H., 1968. Some morphological aspects of active sodiumtransport. The epithelium of the frog skin. J. Cell Biol. 36, 625–638.

Warburg, M.R., 1995. Hormonal effect on the osmotic, electrolyte and nitrogenbalance in terrestrial amphibia. Zool. Sci. 12, 1–11.

Willumusen, J.N., Amstrup, J., Mobjerg, N., Jespersen, A., Kristensen, P.,Larsen, E.H., 2002. Mitochondria-rich cells as experimental model instudies of epithelial chloride channels. Biochim. Biophys. Acta 1566,28–43.

Word, J.M., Hillman, S.S., 2005. Osmotically absorbed water preferentiallyenters the cutaneous capillaries of the pelvic patch in the toad Bufo marinus.Physiol. Biochem. Zool. 78, 40–47.

Yang, B., Gillespie, A., Carlson, E.J., Epstein, C.J., Verkman, A.S., 2001.Neonatal mortality in an aquaporin-2 knock-in mouse model of recessivenephrogenic diabetes insipidus. J. Biol. Chem. 276, 2775–2779.

Yasui, M., 2004. Molecular mechanisms and drug development in aquaporinwater channel diseases: structure and function of aquaporins. J. Pharm. Sci.96, 260–263.

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