Amphibian aquaporins and adaptation to terrestrial environments: a review

In many anurans, the pelvic patch of the ventral skin and the urinary bladder are important osmoregulatory organs. Since the discovery of water channel 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, and 5, 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 by immunohistochemistry. Tree frog type-a2 AQPs, AQP-h2 and AQP-h3, are vasotocin-regulated water channels predominant in the osmoregulatory organs. 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 the cytoplasmic pool to the apical plasma membrane of the granular cells. On the other hand, type-1 AQPs, Rana FA-CHIP and Hyla AQP-h1, are detected at the endothelial cells of blood capillaries in frog osmoregulatory organs. These findings suggest that AQP-h2 and AQP-h3 are key players for transepithelial water movement, and that FA-CHIP and AQP-h1 might be important for the transport of absorbed water into the blood flow. Comparative investigation of type-a2 AQPs in anurans further revealed that AQP-h2 and -h3-like molecules might exist at the urinary bladder and the pelvic skin, respectively, in various anurans from aquatic species to arboreal dwellers. AQP-h2-like protein is also detected in the pelvic skin of terrestrial and arboreal species. It is possible that this molecule might have occurred in the pelvic skin as anurans penetrated into drier environments.     

Cutaneous blood flow and water absorption by dehydrated toads

Blood cell flux (BCF) in ventral pelvic skin capillaries was measured in toads, Bufo woodhouseii and Bufo punctatus, using a chamber that allowed hydration behavior and water absorption to be observed concurrently in unrestrained animals. Dehydrated B. woodhouseii and B. punctatus placed on a rehydration solution significantly increased BCF relative to that on a dry surface in less than 2 min. Skin contact with a rehydration solution rather than dehydration alone is the primary stimulus for increased seat patch blood flow. In B. woodhouseii, the water absorption response was initiated after the increase in BCF had started but before maximum BCF was reached. BCF and water uptake across the ventral skin of both species placed on deionized water were not different from those of toads placed on 50 mM NaCl. Similarly, no significant correlation between BCF and rate of water uptake could be observed in dehydrated toads of either species. Angiotensin II (AII) injection in hydrated B. punctatus had no effect on BCF, suggesting that factors other than AII are responsible for the increase in blood flow upon water contact in dehydrated toads.     

Water potential receptors in the skin regulate blood perfusion in the ventral pelvic patch of toads

Blood cell flux (BCF) in ventral pelvic skin capillaries was measured in conscious unrestrained Bufo bufo, using a laser Doppler flowcytometer. Hydrated toads responded to water contact with a small but significant increase in BCF. Dehydration alone did not change the BCF in seat patch skin before water contact. However, water contact by dehydrated toads elicited a rapid 600% increase in BCF. The BCF and water uptake of dehydrated toads rehydrating in water declined over 2 h but remained significantly above the low, constant values measured in hydrated toads. Arginine vasotocin injection in hydrated toads did not change skin BCF, but water uptake increased, and urine production decreased. Injection of the beta -adrenergic agonist isoproterenol increased BCF in hydrated toads by 900% and also increased the rate of water uptake. These increases corresponded in magnitude and duration to the response to water contact observed in dehydrated toads. Injection of dehydrated toads with the beta -adrenergic antagonist propranolol significantly reduced both BCF and water uptake. These results are consistent with an autonomic reflex mediated by skin water potential receptors that regulate blood perfusion of ventral pelvic skin.     

Spatial and Temporal Expression of the Ventral Pelvic Skin Aquaporins duringMetamorphosis of the Tree Frog, <Emphasis Type="Italic">Hyla Japonica</Emphasis>

Most adult anurans absorb water through their ventral skin to maintain the proper water balance. We examined spatial and temporal expression of frog (Hyla japonica) aquaporins, Hyla AQP-h2 and AQP-h3 proteins, in the ventral pelvic skin by using specific antibodies. Immunofluorescence indicates that AQP-h2 and AQP-h3 first appear in the granular cells of the pelvic skin of the tadpoles at Gosner stage 42, and such labeling is seen in later stages as well. These findings were confirmed by Western blot analysis. In addition, Northern blot analysis demonstrated that V₂-type vasotocin (AVT)-receptor mRNA is first expressed at the same stage as are the AQP proteins, which suggests a functional relationship between expression of AQP proteins and AVT receptor. Also, AQP expression in the ventral pelvic skin is consistent with the morphological changes that occur in the skin for adaptation from life in water to that on land.This revised version was published online in August 2005 with a corrected cover date.     

Comparison of dehydration and angiotensin II-stimulated cutaneous drinking in toads, Bufo punctatus

Toads (Bufo punctatus) use a sequence of two postures to place the ventral skin on a moist surface and absorb water osmotically. First, the skin contacts the surface (seat patch down, SPD), and then the hindlimbs are abducted to maximize skin contact area (water absorption response, WR). Toads modulated behavior in response to hydration status and osmotic content of the hydration source. Dehydrated toads placed on water displayed both SPD and WR. Hydrated toads injected with angiotensin II (AII) displayed SPD longer than Ringer-injected controls but did not initiate WR and absorbed less water than dehydrated toads. These results suggest that dehydration has a more robust dipsogenic effect than AII. Dehydrated toads placed on 250 mM NaCl briefly initiated SPD but not WR. The addition of amiloride to the hyperosmotic salt solution resulted in brief display of WR but no water loss. Hydrated toads placed on 250 mM NaCl showed shorter periods of SPD behavior. The combination of AII injection and amiloride addition to the salt solution increased SPD initiation but SPD duration was short and water loss was prevented. Neither AII nor dehydration overrides chemosensory mechanisms in the skin that suppress cutaneous drinking from hypertonic solutions.     

Gene cloning and expression of an aquaporin (AQP-h3BL) in the basolateral membrane of water-permeable epithelial cells in osmoregulatory organs of the tree frog

An aquaporin (Hyla AQP-h3BL), consisting of 292 amino acid residues, has been cloned from the urinary bladder of Hyla japonica. In a swelling assay using Xenopus oocytes, AQP-h3BL cRNA-injected oocytes developed a sevenfold and 2.8-fold higher permeability to water and glycerol, respectively, than the water-injected oocytes. This permeability was inhibited by HgCl2. Immunofluorescence revealed that AQP-h3BL is localized in the basolateral plasma membrane of both granular cells in the ventral pelvic and dorsal skins and the secretory cells in the mucous glands. Immunopositive cells were also observed in the basolateral membrane of principal cells in the collecting ducts and in a portion of the late distal tubules in the kidneys, as well as in the principal cells of the urinary bladder. Sequence homology suggests that AQP-h3BL is a homolog to mammalian AQP3. This conclusion is supported by the observed localization of AQP-h3BL to the basolateral membrane in water- and glycerol-permeable epithelial cells. In ventral pelvic skins and urinary bladders, water enters into the cytoplasm through the apical plasma membrane at sites where AQP-h2, sometimes in association with AQP-h3, responds to stimulation by vasotocin; the water exits throughout AQP-h3BL to extracellular spaces. In the mucous glands, on the other hand, water enters throughout this AQP-h3BL and exits through AQP-x5, which is in the apical membrane of secretory cells. Thus, water homeostasis in the frog body is regulated by AQP-h3BL expressed in the basolateral membrane in concert with arginine vasotocin (AVT)-dependent or AVT-independent AQP.     

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.     

Vascular aspects of water uptake mechanisms in the toad skin: perfusion, diffusion, confusion

Blood cell flow (BCF) in the water absorbing "seat patch" region of toad skin was measured with laser Doppler flow cytometry. BCF of dehydrated toads increased by a factor of 6-8 when water contact was made and declined gradually as toads rehydrated. Water absorption was initially stimulated and declined in parallel with BCF. Water absorption measured during the initial rehydration period did not correlate with BCF and hydrated toads injected with AVT increased water absorption without an increase in BCF indicating the lack of an obligate relation between blood flow and water absorption. Aquaporins 1-3 were characterized by RT-PCR analysis of seat patch skin. AQP 1 was localized in the endothelium of subepidermal capillaries and serves as a pathway for water absorption in series with the apical and basolateral membranes of the epithelium. Dehydrated toads rehydrated more rapidly from dilute NaCl solutions than from deionized water despite the reduced osmotic gradient. BCF of toads rehydrating on 50 mM NaCl was not different than on deionized water and blocking Na+ transport with 100 microM amiloride did not reduce water absorption from 50 mM NaCl. Thus, neither circulation nor solute coupling explains the greater absorption from dilute salt solutions. Rehydration from 10 mM CaCl2 was stimulated above that of DI water by a similar degree as with 50 mM NaCl suggesting the anion might control water permeability of the skin.     

Comparative effects of angiotensin II on osmotic water permeability in the toad (Bufo arenarum)

The possible relationship between the renin-angiotensin system and water balance in the toad Bufo arenarum has been indirectly explored. A positive correlation was found between the hydrosmotic response of ventral pelvic toad skin to angiotensin II (A II) and some age indicators (body weight, snout-urostyle length or head width). A different hydrosmotic response for oxytocin and isoproterenol (but not for A II) was found between four cutaneous regions of toad body. We conclude that A II may not be directly involved in the regulation of water balance mediated by water absorption across the skin of Bufo arenarum toads.     

Molecular systematics of African 20-chromosome toads (Anura: Bufonidae)

Intrageneric lineages and the historical biogeography of toads, genus Bufo, are poorly resolved due to their conservative morphology, their highly conserved karyotypes (typically 2N = 22), and erratic patterns of interspecific hybridisation. Here, we use mitochondrial and nuclear DNA sequence data to reconstruct relationships of the 20-chromosome toads, a major component of the African bufonid fauna. Mitochondrial 12S and 16S sequences from 29 species revealed two independent transitions between phenotypically distinct savannah and forest adapted forms. Analyses of mitochondrial 12S, 16S, and ND2, along with nuclear ACTC and Rhodopsin sequences from 12 species greatly increased bootstrap, and likelihood support for internal branches including a basal split into two pan-African 20-chromosome clades. Hybridisation is a weak indicator of phylogenetic relationship as it occurs across these deeply divergent clades, between Bufo rangeri and Bufo gutturalis. These analyses suggest a secondary reversion to 22-chromosomes in Bufo pardalis, within the 20-chromosome group, although we could not reject an alternative hypothesis that this lineage forms a sister to all 2N = 20 toads. Other informally recognised 22-chromosome groups form independent phylogenetic lineages outside the 20-chromosome group, such as the Angusticeps and Vertebralis divisions. Bufo lindneri, from the Taitanus division, is closely related to Stephopaedes anotis, and these species should be considered congeners.     

Mortality of captive Canadian toads from Basidiobolus ranarum mycotic dermatitis

Twenty-six adult free-ranging Canadian toads (Bufo hemiophrys) were collected from northeastern North Dakota (USA) during the last week of August 1994 and placed in captivity. During late December and January 1995, 21 Canadian toads died. Clinical signs included increased time sitting in water bowls, darkened dorsal skin, constant arching of their backs, and hyperemia and sloughing of ventral epidermis. The condition progressively worsened until death occurred within 5 to 7 days after onset of clinical disease. Mycotic dermatitis due to Basidiobolus ranarum was diagnosed in all toads and the fungus was isolated from 11 (52%) of 21 toads. Histology of the ventral skin and digits revealed numerous fungal spherules and occasional hyphae without significant inflammatory reaction. This condition clinically resembled red leg associated with Aeromonas hydrophila and many other bacterial organisms, and the diseases could be confused without appropriate diagnostic tests. This also is the first report of B. ranarum causing clinical disease in a toad species.     

Immunocytochemical studies on translocation of phosphorylated aquaporin-h2 protein in granular cells of the frog urinary bladder before and after stimulation with vasotocin

We have generated a specific antibody against phosphorylated aquaporin-h2 (pAQP-h2) protein to investigate the role of phosphorylation in the translocation of AQP-h2 protein within the granule cells of the urinary bladder of the frog (Hyla japonica). The antibody was generated against a synthetic peptide (ST-160) corresponding to amino acids 255–268, with a phosphorylated Ser-262, a residue that is putatively phosphorylated by protein A kinase. Using this antibody, we found, by Western blot analysis, that phosphorylation of the AQP-h2 protein rapidly increased within 2 min after vasotocin (AVT) stimulation and remained at a higher than normal level for 15 min. Moreover, quantitative immunoelectron microscopy indicated that the location of the AQP-h2 protein dramatically changed after AVT stimulation. Before stimulation, pAQP-h2 protein was localized in only a small number of intracellular vesicles near the nucleus of the granular cells, whereas the labeling density of the intracellular vesicles and the apical membrane rapidly increased after stimulation. This finding was also confirmed by the results of an immunofluorescence study. Thus, phosphorylation of AQP-h2 protein seems to be essential for translocation of the protein from the cytoplasmic pool to the apical plasma membrane of the granular cells in frog urinary bladder. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan to S.T.     

Dermatitis in captive Wyoming toads (Bufo baxteri) associated with Fusarium spp

From May 2007 to June 2008, 30 of 49 Wyoming toads (Bufo baxteri) kept at Omaha's Henry Doorly Zoo (Nebraska, USA) died showing clinical signs of ventral erythema, inappetance, lethargy, and delayed righting reflex. Treatment with antifungals and antibiotics was unsuccessful in all cases. Histopathologic analyses revealed dermatitis as the primary problem in 20 of 21 toads in which skin was examined. Fungal dermatitis was present in 17 toads, with hyphae approximately 1-3 μm in diameter, and parallel cell walls and frequent septations. In 14 animals, the fungal dermatitis was the main pathologic lesion. Several species of bacteria were associated with all cases. A few animals tested positive for Ranavirus using polymerase chain reaction. Fusarium sp. was consistently cultured from skin, feces, kidneys, and from powdered food provided to crickets. Four isolates were identified as Fusarium proliferatum, Fusarium oxysporum, Fusarium solani, and Fusarium verticillioides, which suggested a secondary role of fungi. A specific underlying cause of disease could not be found, although the roles of humidity and Ranavirus infection are discussed, along with the well-known susceptibility of bufonids to fungal dermatitis.     

Evidence of nitric oxide and angiotensin II regulation of circulation and cutaneous drinking in Bufo marinus

The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (l-NAME) increased vascular resistance (VR) 10% above baseline of 3.08+/-0.08 (n=11) mmHg/mL/min at 10 mg/kg and 20% above 3.05+/-0.08 (n=9) at 50 mg/kg in anesthetized toads (Bufo marinus). Blood pressure was unaffected by either dose of L-NAME. Blood flow decreased at the higher dose of L-NAME. L-arginine (300 mg/kg) reversed the effects of L-NAME on VR and blood flow in toads treated with 10 mg/kg but not with 50 mg/kg. Injection of 50 mg/kg L-NAME into empty-bladder toads produced a 10% decrease in water uptake, J(v), resulting in a J(v) of 1,267+/-11 cm(3)/cm(2)/s x 10(-7) (n=9) compared to 1,385+/-12 (n=8) for controls. Injection of 10 microg/kg angiotensin II (ANG II) increased J(v) 15% across the pelvic patch (J(v), cm(3)/cm(2)/s x 10(-7)), resulting in a J(v) of 1,723+/-12 cm(3)/cm(2)/s x 10(-7) (n=8) compared to 1,471+/-12 (n=8) for controls. It is hypothesized that during cutaneous drinking blood flow into the capillary bed of the pelvic patch is regulated by nitric oxide and ANG II.     

Expression of a mammalian aquaporin 3 homolog in the anterior pituitary gonadotrophs of the tree frog, <Emphasis Type="Italic">Hyla japonica</Emphasis>

Aquaporins (AQPs) are a family of water channel proteins that play a major role in maintaining water homeostasis in various organisms. Several AQPs have been identified in the tree frog, Hyla japonica. Of these, AQP-h3BL, which is expressed in the basolateral membrane of the epithelial cells, is a homolog of mammalian AQP3. Using immunohistochemistry and in situ RT-PCR, we have demonstrated that AQP-h3BL is expressed in the anterior pituitary gonadotrophs of the tree frog but not in the other hormone-producing cells of the anterior pituitary. In gonadotrophs labeled for luteinizing hormone subunit-β (LHβ), AQP-h3BL protein was found to reside in the plasma membrane, the nuclear membrane and the cytoplasm. Double-labeling of AQP-h3BL mRNA and LHβ protein revealed that AQP-h3BL mRNA is expressed in the gonadotrophs. Following stimulation by gonadotropin-releasing hormone (GnRH), the label for AQP-h3BL localized in the plasma membrane became more intense, concomitant with the transport of LHβ-positive materials to the plasma membrane. These developments coincided with a decrease in the labeling density in the cytoplasm and near the nuclear membrane, suggesting that the latter localizations may function as “storage area“ for AQP-h3BL. Immunoelectron microscopy also confirmed these localizations of AQP-h3BL protein. Based on these results, we suggest that AQP-h3BL protein in the frog gonadotrophs is involved in the formation of secretory granules, the swelling and increase in the volume of the granules and exocytosis.     

Basal and amiloride-induced short-circuit current across isolated toad skin (Bufo arenarum)

We have previously demonstrated that amiloride (amil) addition to the isolated ventral pelvic (VPel) skin of Bufo arenarum toad induces negative short-circuit current values, which are equivalent to the isotopically measured net chloride transport. In the present work, we found that exposure of various regions of toad skin to amil yielded different values of short-circuit current (aSCC): negative aSCC was found in the VPel and ventral pectoral skin, while those of the dorsal one were not different from zero. The distinct values of aSCC found show a regional difference in the active chloride absorption, probably related to postural adaptations. A possible role of this adaptation would be related to chloride participation in the saline balance of the animals, or the maintenance of epithelial integrity.     

中药蟾酥的药理作用研究进展

蟾酥是我国传统中药,是蟾蜍科动物中华大蟾蜍或黑眶蟾蜍的干燥分泌物。蟾酥所含化学成分主要有蟾蜍内酯类、蟾毒色胺类等,现代药理学研究表明其具有抗肿瘤、强心、局麻、镇痛、抗炎等多种作用。本文综述近年来国内外学者对蟾酥的化学成分、药理作用的研究概况。     

The spinal nerves innervate putative chemosensory cells in the ventral skin of desert toads, Bufo alvarius

Toads normally obtain water by absorption across their skin from osmotically dilute sources. When hyperosmotic salt solutions are presented as a hydration source to dehydrated desert toads, they place the ventral skin onto the source but soon afterwards escape to avoid dehydration. The escape behavior coincides with neural excitation of the spinal nerves that innervate putative chemosensory cells in the ventral skin. In the present study, fluorescent dye translocated through the spinal nerves to those receptor cells in the epidermis was photoconverted in the presence of 3, 3'-diaminobenzidine tetrahydrochloride for electron-microscopic observation of the cells and associated nerve terminals. Most of the photoconverted cells were located in the deepest layer of the epidermis, with some being in more intermediate layers. No labeled cell was seen in the outermost layer of living cells. In desert toads, flask cells and Merkel cells are occasionally seen in the epidermis. An association of nerve fibers with these epidermal cells has been reported in some species of the anurans. In the present study, however, the cytological features of the photoconverted cells are neither reminiscent of flask cells nor Merkel cells, but are similar to those of surrounding epithelial cells in each layer of the epidermis. We hypothesize a sensory function for these cells, because they have a close association with nerve fibers and participate in the transepithelial transport of salts that must pass through all cell layers of the skin.     

Phylogeny of South American Bufo (Anura: Bufonidae) inferred from combined evidence

Despite the considerable research that has focused on the evolutionary relationships and biogeography of the genus Bufo, an evolutionary synthesis of the entire group has not yet emerged. In the present study, almost 4 kb of DNA sequence data from mitochondrial (12S, tRNA^Val, and 16S) and nuclear (POMC; Rag-1) genes, and 83 characters from morphology were analysed to infer a phylogeny of South American toads. Phylogenies were reconstructed with parsimony and maximum likelihood and Bayesian model-based methods. The results of the analysis of morphological data support the hypothesis that within Bufo , some skull characters (e.g. frontoparietal width), correlated with the amount of cranial ossification, are prone to homoplasy. Unique and unreversed morphological synapomorphies are presented that can be used to diagnose recognized species groups of South American toads. The results of all phylogenetic analyses support the monophyly of most species groups of South American Bufo . In most DNA-only and combined analyses, the South American (minus the B. guttatus and part of the ' B. spinulosus ' groups), North American, Central American, and African lineages form generally well-supported clades: ((((((((South America) (North America + Central America)) Eurasia) Africa) Eurasia) South America) West Indies) South America). This result confirms and extends prior studies recovering South American Bufo as polyphyletic. The biogeographical results indicate that: (1) The origin of Bufo predates the fragmentation of Gondwana; (2) Central and North American species compose the sister group to a large, 'derived' clade of South American Bufo ; and (3) Eurasian species form the sister group to the New World clade.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 146 , 407–452.     

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus)

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.