Metabolic depression and Na+/K+ gradients in the aestivating Australian goldfields frog,Neobatrachus wilsmorei

During aestivation the metabolic rate of the Australian goldfields frog Neobatrachus wilsmorei was reduced by 80% from its standard metabolic rate. The in vitro rate of oxygen consumption of isolated muscle and skin from aestivating frogs was up to 50% lower than that of the non-aestivating frogs. This in vitro rate of oxygen consumption was maintained for 6–12 h, indicating an intrinsic metabolic depression of tissues during aestivation. Frogs became dehydrated during aestivation. Muscle, skin and liver also became dehydrated during aestivation, but brain and kidney did not. Na⁺ and K⁺ contents and extracellular space measurement for muscle indicated that ion gradients were maintained across the muscle cell membrane during aestivation. Increases in plasma concentrations of Na⁺ and K⁺ were matched with similar increases in muscle intracellular ion concentrations. Extracellular space measurements were unsuccessful in the other tissues, but K⁺ content in all tissues (per dry weight) was maintained during aestivation, and the concentration of plasma K⁺ did not increase above that which can be accounted for by dehydration, indicating that K⁺ gradients were maintained.Abbreviations bm body mass - DPM disintegrations per minute - dw dry weight - MR metabolic rate - vO₂ rate of oxygen consumption - ww wet weight     

Metabolic depression during aestivation does not involve remodelling of membrane fatty acids in two Australian frogs

Changes in membrane lipid composition (membrane remodelling) have been associated with metabolic depression in some aestivating snails but has not been studied in aestivating frogs. This study examined the membrane phospholipid composition of two Australian aestivating frog species Cyclorana alboguttata and Cyclorana australis. The results showed no major membrane remodelling of tissue in either frog species, or in mitochondria of C. alboguttata due to aestivation. Mitochondrial membrane remodelling was not investigated in C. australis. Where investigated in C. alboguttata, total protein and phospholipid content, and citrate synthase (CS) and cytochrome c oxidase (CCO) activities in tissues and mitochondria mostly did not change with aestivation in liver. In skeletal muscle, however, CS and CCO activities, mitochondrial and tissue phospholipids, and mitochondrial protein decreased with aestivation. These decreases in muscle indicate that skeletal muscle mitochondrial content may decrease during aestivation. Na⁺K⁺ATPase activity of both frog species showed no effect of aestivation. In C. alboguttata different fat diets had a major effect on both tissue and mitochondrial phospholipid composition indicating an ability to remodel membrane composition that is not utilised in aestivation. Therefore, changes in lipid composition associated with some aestivating snails do not occur during aestivation in these Australian frogs.     

Metabolic depression and sodium-potassium ATPase in the aestivating frog, Neobatrachus kunapalari

In aestivation the metabolic rate of the Australian desert frog Neobatrachus kunapalari was 50–67% lower than in the non-aestivating state. The rate of O₂ consumption of isolated muscle, skin and brain was measured in both metabolic states. The average rate of O₂ consumption of muscle was 30% lower and brain 50% lower in aestivating frogs, while the rate of O₂ consumption of skin was the same. The reduction in muscle could account for a large proportion of whole animal metabolic depression. To look for evidence of a reduction in energy demand in the tissues we measured the ouabain-sensitive fraction of tissue rate of O₂ consumption, which is considered to be the proportion of metabolism used for transmembrane Na⁺/K⁺ pumping. Ouabain inhibited the in vitro rate of O₂ consumption of skin by a average of 20% and of brain by an average of 30%. However, in muscle, ouabain stimulated in vitro O₂ consumption. Despite the 50% reduction in the in vitro rate of O₂ consumption of brain during aestivation, neither the ouabain-sensitive nor ouabain-insensitive fractions were found be statistically different, possibly because of the large individual variation in the degree of ouabain inhibition. A reduction in the level of ion pumping during aestivation was therefore not demonstrated in any tissue. Measurement of the level of the enzyme Na⁺K⁺-ATPase in skeletal muscle, ventricle, kidney and brain showed that there was no change in the amount of this enzyme in the aestivating frogs. Measurement of the levels of adenylates in muscle and liver showed that the adenylate energy charge was maintained in aestivation, but that there was a reduction in ATP in liver and a reduction in the level of total adenylates in both tissues, which could be an adaptation of the tissues to a lower energy turnover. Accepted: 22 July 1996     

Effect of aestivation on muscle characteristics and locomotor performance in the Green-striped burrowing frog, Cyclorana alboguttata

The Green-striped burrowing frog, Cyclorana alboguttata survives extended drought periods by burrowing underground and aestivating. These frogs remain immobile within cocoons of shed skin and mucus during aestivation and emerge from their burrows upon heavy rains to feed and reproduce. Extended periods of immobilisation in mammals typically result in muscle atrophy and a decrease in muscle performance. We examined the effect of aestivation and hence prolonged immobilisation, on skeletal muscle mass, in vitro muscle performance, and locomotor performance in C. alboguttata. Frogs were aestivated in soil for 3 months and were compared with control animals that remained active, were fed, and had a continual supply of water. Compared to the controls, the wet mass of the gastrocnemius, sartorius, gracilus major, semimembranosus, peroneus, extensor cruris, tibialis posticus and tibialis anticus longus of aestivators remained unchanged indicating no muscle atrophy. The in-vitro performance characteristics of the gastrocnemius muscle were maintained and burst swimming speed was unaffected, requiring no recovery from the extended period of immobilisation associated with aestivation. This preservation of muscle size, contractile condition and locomotor performance through aestivation enables C. alboguttata to compress their life history into unpredictable windows of opportunity, whenever heavy rains occur.     

Water balance and arginine vasotocin in the cocooning frog Cyclorana platycephala (hylidae)

It is well established that forming a cocoon, for frog species capable of doing so, markedly reduces evaporative water loss; however, the capacity of cocooned frogs to maintain hydration during extended estivation is not well understood. The combined effects of long-term estivation and water loss were examined in the cocoon-forming species Cyclorana platycephala by assessing the hydration state of the frogs throughout a 15-mo estivation period. Frogs lost mass throughout the 15-mo period to a maximum of 36%+/-6.5% of their initial standard mass. Plasma osmolality reached maximal levels by the ninth month of estivation at 487 mOsm kg(-1) and then remained stable to the fifteenth month of estivation. Urine osmolality continued to increase to the fifteenth month of estivation, at which point plasma and urine concentrations were isosmotic. The use of bladder water to counter losses from circulation was indicated by the relatively slow rate of increase in plasma osmolality with mass loss and the progressive increase in urine osmolality. For estivating frogs, evidence was found for a possible threshold relationship between plasma osmolality and plasma arginine vasotocin (AVT) concentration. After estivation, plasma AVT concentrations decreased markedly after 15-mo estivators were placed in water for 2 h, suggesting that high levels of AVT may not be integral to rapid rehydration in this species.     

Immunoreactive Tamm-Horsfall protein in the kidney and skin of the frog Rana temporaria

Summary Tamm-Horsfall protein (THP) is the main protein in normal human urine, and is found in the thick limb of the Loop of Henle in human kidney, and in other mammalian species. The skin of the frog, Rana temporaria, has similar physiological properties to this mammalian kidney tissue. In the present study, an immunohistological method involving an antibody to human THP was used to investigate the distribution of this distinctive protein in frog kidney and skin, and to compare its distribution with that found in the kidney tubules of rat and rabbit. THP-positive material was detected in the distal renal tubules and nephric duct of frogs, and was also located in the superificial epidermis of skin. It is suggested that its presence in amphibian skin is consistent with the hypothesis that THP is an important component of tissues that absorb sodium and chloride ions, but remain impermeable to water.     

Integumentary structure and its relationship to wiping behaviour in the common Indian tree frog, Polypedates maculatus

Skin of the Indian tree frog, Polypedates maculatus (Rhacophoridae), was studied in the context of self-wiping behaviour which functions to expel and distribute cutaneous secretions recently shown to retard evaporative water loss. The secretions contain both mucus and lipids and are derived from a common gland considered to be homologous with characteristic anuran mucous glands. The glands are bipotent and secrete both mucus and lipoid products which are evidently mixed within the glandular lumen. Another type of gland resembling characteristic anuran serous (or granular) glands is found in dorsal but not ventral skin, whereas the lipid-secreting mucous glands are found in skin associated with all body surfaces. There is no distinct, lipid-secreting gland present in the skin of this species other than the mucous glands. These histochemical data complement the earlier finding that resistance to evaporative water loss in this species is relatively small compared with phyllomedusine 'waterproof frogs which also exhibit wiping behaviour associated with secretion of lipids. Thus, wiping behaviour may have evolved in association with mucous secretions before dominant lipoid secretions resulted from strong selection for water conservation.     

Metabolic depression during aestivation in Cyclorana alboguttata

The green striped burrowing frog, Cyclorana alboguttata, spends, on average, nine to ten months of every year in aestivation. Recently, C. alboguttata has been the focus of much investigation regarding the physiological processes involved in aestivation, yet our understanding of this frog's capacity to metabolically depress remains limited. This study aimed to extend our current knowledge of metabolic depression during aestivation in C. alboguttata. C. alboguttata reduced whole animal metabolism by 82% within 5 weeks of aestivation. The effects of aestivation on mass specific in vitro tissue metabolic rate (VO₂) varied among individual organs, with muscle and liver slices showing significant reductions in metabolism; kidney VO₂ was elevated and there was no change in the VO₂ of small intestine tissue slices. Organ size was also affected by aestivation, with significant reductions in the mass of all tissues, except the gastrocnemius. These reductions in organ size, combined with changes in mass specific VO₂ of tissue slices, resulted in further energy savings to aestivating animals. This study shows that C. alboguttata is capable of selectively down- or up-regulating individual tissues, using both changes in metabolic rate and morphology. This strategy allows maximal energy savings during aestivation without compromising organ functionality and survival at arousal.     

Changes in body fluids of the cocooning fossorial frog Cyclorana australis in a seasonally dry environment

We investigated changes in the lymph (equivalent to plasma) and urine of the cocooning frog Cyclorana australis during the dry season in monsoonal northern Australia. Frogs in moist soil for two days were fully hydrated (lymph 220 mOsm kg(-1), urine 49 mOsm kg(-1)). From five weeks onwards the soil was dry (matric potential <-8000 kPa). Aestivating frogs at three and five months formed cocoons in shallow (<20 cm) burrows and retained bladder fluid (25-80% of standard mass). After three months, urine but not lymph osmolality was elevated. After five months, lymph (314 mOsm kg(-1)) and urine (294 mOsm kg(-1)) osmolality and urea concentrations were elevated. Urea was a major contributing osmolyte in urine and accumulated in lymph after five months. Lymph sodium concentration did not change with time, whereas potassium increased in urine after five months. Active animals had moderate lymph osmolality (252 mOsm kg(-1)), but urea concentrations remained low. Urine was highly variable in active frogs, suggesting that they tolerate variation in hydration state. Despite prolonged periods in dry soil, osmolality increase in C. australis was not severe. Aestivation in a cocoon facilitates survival in shallow burrows, but such a strategy may only be effective in environments with seasonally reliable rainfall.     

NaCl adaptation in Rana ridibunda and a comparison with the euryhaline toad Bufo viridis.

The physiological adaptation of the frog Rana ridibunda to saline environment was studied. It was found that blood was always hypertonic to the external solution, but at the highest salinity tolerated (i.e. 300 mOsM) the osmotic gradient across the skin was nearly abolished. Water uptake by the living frog remained unchanged, whereas sodium transport across the skin decreased markedly. Neurohypophyseal hormone increased water uptake and sodium transport to levels similar to those in tap water frogs. Water content of the tissues was not affected by saline adaptation, although it varied appreciably under acute conditions. Oxygen consumption increased in dehydrated frogs, but not in adapted ones. The results are discussed and compared to the euryhaline toad Bufo viridis; the importance of high urea levels for high salt adaptation is stressed.     

Comparison of ACE activity in amphibian tissues: Rana esculenta and Xenopus laevis

Angiotensin converting enzyme (ACE) is the dipeptidyl-carboxypeptidase of the renin-angiotensin system involved in the control of blood pressure and hydromineral metabolism. It converts angiotensin I to angiotensin II, the biologically active octapeptide. Angiotensin converting enzyme-like activity has been demonstrated in a wide range of vertebrates. The presence of ACE was investigated in tissues of two amphibian species, the frog Rana esculenta and the toad Xenopus laevis. ACE activities were determined by specific substrate hydrolysis in gut, gonads, lung, kidney, heart, liver, skin, erythrocytes, and muscle homogenates and plasma by means of high performance liquid chromatography. Significant ACE activity was found in gut, gonads, lung and kidney, while that in heart, liver, skin, erythrocytes, muscle, and plasma was very low. Testis of toad contained the highest ACE activity, while that in erythrocytes of male and female frogs was notable.     

Storage excretion in the Indian apple snail,Pila globosa (Swainson), during aestivation

Uric acid accumulates in several tissues of the Indian apple snail, P. globosa, during aestivation. This accumulation is particularly high in the foot muscle and reproductive organs. Since the kidney, a tiny organ in the snail, has only a limited capacity to store uric acid, extrarenal tissues are used as storage depots of uric acid during aestivation. It is suggested that the aestivating snail, faced with a cleidoic situation, resorts to storage excretion, a phenomenon well documented in insects.     

Ultrastructural and histochemical study on gills and skin of the Senegal sole, Solea senegalensis

This study was undertaken to identify the normal ultrastructural features of gills and skin of the Senegal sole, Solea senegalensis, for a comparative measure to morphological alterations caused by environmental stressors such as reduced water quality and diseases. In the Senegal sole skin, four morphologically distinct layers were identified: cuticle, epidermis, dermis and hypodermis. The epidermis was composed of stratified epithelium containing three cellular layers: the outermost or mucosa layer, the middle or fusiform layer and the stratum germinativum or the basal layer. In the mucosa, two mucous cell types were differentiated: type A cells containing several round vesicles of different electron density and type B cells containing mucosomes of uniform electron density. Senegal sole have five pairs of gill arches, each containing two rows of well‐developed and compactly organized primary filaments and secondary lamellae. Fingerprint‐like microridges were observed on the surface of epithelial cells. The branchial lamellae epithelium consisted of different cell types: pavement, mucous and chloride. Between the chloride cells and the larger pavement cells, accessory cells were observed. Complexes of tight junctions and desmosomes were frequently observed between adjacent chloride and epithelial cells. Neutral mucosubstances and/or glycoconjugates were observed in the epidermis, dermis and hypodermis of S. senegalensis skin. Proteins rich in different amino acids, such as arginine and cysteine, reacted negatively or weakly positive in the epidermis, dermis and hypodermis. In gills, some mucous cells responded weakly positive to periodic acid‐Schiff (PAS) reaction but were strongly stained with Alcian Blue at pH 0.5, 1 and 2.5. When Alcian Blue pH 2.5–PAS reaction was performed, most mucous cells were stained blue (carboxylated mucins) and some mucocytes stained purple, indicating a combination of neutral and acid mucins. Proteins rich in cysteine‐bound sulphydryl (‐SH‐) and cystine disulphide (‐S‐S‐) groups were strongly detected in branchial and epidermal mucous cells, whereas lysine, tyrosine and arginine containing proteins showed very weak staining in both epidermal and branchial mucous cells. Protein reactions were strongly positive in the pillar cells, except for those rich in tryptophan, whereas the branchial cartilaginous tissue did not show an important reaction. The performed lipid reactions were negative in goblet and chloride cells. It is concluded from this study that ultrastructural and cytohistochemical features of the Senegal sole skin and gills may serve as control structures in both natural and aquaculture systems to monitor or detect environmental stress responses at the histological level.     

Central angiotensin II stimulates cutaneous water intake behavior via an angiotensin II type-1 receptor pathway in the Japanese tree frog Hyla japonica

Angiotensin II (Ang II) stimulates oral water intake by causing thirst in all terrestrial vertebrates except anurans. Anuran amphibians do not drink orally but absorb water osmotically through ventral skin. In this study, we examined the role of Ang II on the regulation of water-absorption behavior in the Japanese tree frog (Hyla japonica). In fully hydrated frogs, intracerebroventricular (ICV) and intralymphatic sac (ILS) injection of Ang II significantly extended the residence time of water in a dose-dependent manner. Ang II-dependent water uptake was inhibited by ICV pretreatment with an angiotensin II type-1 (AT₁) receptor antagonist but not a type-2 (AT₂) receptor antagonist. These results suggest that Ang II stimulates water-absorption behavior in the tree frog via an AT₁-like but not AT₂-like receptor. We then cloned and characterized cDNA of the tree frog AT₁ receptor from the brain. The tree frog AT₁ receptor cDNA encodes a 361 amino acid residue protein, which is 87% identical to the toad (Bufo marinus) AT₁ receptor and exhibits the functional characteristics of an Ang II receptor. AT₁ receptor mRNAs were found to be present in a number of tissues including brain (especially in the diencephalon), lung, large intestine, kidney and ventral pelvic skin. When tree frogs were exposed to dehydrating conditions, AT₁ receptor mRNA significantly increased in the diencephalon and the rhombencephalon. These data suggest that central Ang II may control water intake behavior via an AT₁ receptor on the diencephalon and rhombencephalon in anuran amphibians and may have implications for water consumption in vertebrates.     

吉林产哈士蟆中氨基酸含量的研究

应用日立835—50型氨基酸自动分析仪,对哈士蟆十种干样品进行了氨基酸成分的比较分析,结果表明:①必需氨基酸含量由高至低依次为雄蛙腿肉,雌蛙腿肉、卵、整只雄蛙、整只雌蛙、雄蛙皮、哈士蟆油、雌蛙皮、雌蛙腿骨、雄蛙腿骨。②氨基酸总量由高至低依次为雄蛙腿肉、雌蛙腿肉、雄蛙皮、雌蛙皮、卵、整只雄蛙、整只雌蛙、哈士蟆油、雌蛙腿骨、雄蛙腿骨。③雄蛙腿肉和雌蛙腿肉中各种氨基酸含量由大到小的排列顺序基本一致。     

Differential NOS expression in freshwater and aestivating Protopterus dolloi (lungfish): heart vs kidney readjustments.

African lungfish Protopterus dolloi is an obligatory air-breather, which aestivates in a cocoon during the dry season. Aestivation associates with functional modifications in many tissues and organs, including heart and kidney. Due to its pleiotropic modulatory effects, nitric oxide (NO), generated by nitric oxide synthases (NOSs), may coordinate organ rearrangement, allowing adaptive adjustments under stressful environmental conditions. By immunofluorescence, Western blotting and NADPH-diaphorase, we examined cardiac and renal localization and activity of NOSs isoforms in both freshwater (FW) and aestivating [6 days (6DA) and 40 days (40DA) of estivation] P. dolloi. In heart and kidney endothelial NOS (eNOS) is the major isoform with respect to inducible and neuronal NOS (iNOS and nNOS, respectively). Cardiac eNOS locates in the epicardium, the trabecular endothelial endocardium, and myocardiocytes of both FW and aestivating fish. Western blotting revealed that cardiac eNOS expression increases in 6DA, but decreases in 40DA fish. In FW fish kidney eNOS is present in vascular endothelial cells and in podocytes of renal corpuscles. In tubular epithelial cells it is restricted to the apical pole. With aestivation, both renal localization and expression of eNOS increase. NADPH-diaphorase revealed an enhancement of cardiac and renal NOS activities during aestivation. Results suggest that in P. dolloi NO contributes, in an autocrine-paracrine fashion, to cardiac and renal readjustments during aestivation. Our findings are of evolutionary interest, since they document for the first time the presence of a NOS system in a ancestral fish, indicative of deep phylogenetic roots of NO bio-synthesis.     

Freeze-Induced Alterations of Translatable mRNA Populations in Wood Frog Organs

To investigate the roles that gene expression and new protein synthesis play in freezing survival by the wood frog, Rana sylvatica, we compared the in vitro translation products made from mRNA isolated from six tissues (liver, brain, heart, muscle, kidney, gut) of control (5 degrees C), frozen (24 h at -2.5 degrees C), and thawed (24 h at 5 degrees C after 24 h frozen) frogs. [(35)S]Methionine-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and located by fluorography. Results indicated specific changes in the translatable populations of mRNA in tissues of freezing-exposed frogs that were largely reversed upon thawing. Differential protein expression was greatest in the comparison of liver from control versus frozen frogs with proteins ranging from 45 to 14.8 kDa identified as enhanced or unique to the frozen state. One unique protein appeared in skeletal muscle (116 kDa) of freeze-exposed frogs while another (52.5 kDa) was enhanced. Analysis of brain and heart each revealed the presence of one protein unique to the frozen state in each (58.9 and 5.9 kDa, respectively) whereas no change in the pattern of in vitro translation products was seen in gut (stomach + intestine combined) or kidney between the three experimental states. These freeze-induced alterations in the populations of translatable mRNA suggest that changes in the complement of specific proteins underlie various adaptive responses that contribute to the freezing survival of this amphibian.     

Frog skin epithelium: electrolyte transport and chytridiomycosis

One unique physiological characteristic of frogs is that their main route for intake of water is across the skin. In these animals, the skin acts in concert with the kidney and urinary bladder to maintain electrolyte homeostasis. Water absorption across the skin is driven by the osmotic gradient that develops as a consequence of solute transport. Our recent study demonstrated that chytridiomycosis, an infection of amphibian skin by the fungal pathogen, Batrachochytrium dendrobatidis, inhibits epithelial Na(+) channels, attenuating Na(+) absorption through the skin. In frogs that become severely affected by this fungus, systemic depletion of Na(+), K(+) and Cl(-) is thought to cause deterioration of cardiac electrical function, leading to cardiac arrest. Here we review the ion transport mechanisms of frog skin, and discuss the effect of chytridiomycosis on these mechanisms.     

Xenopus kidney hyaluronidase-1 (XKH1), a novel type of membrane-bound hyaluronidase solely degrades hyaluronan at neutral pH

In search for Xenopus laevis hyaluronidase genes, a cDNA encoding a putative PH-20-like enzyme was isolated. In the adult frog, this mRNA was only found to be expressed in the kidney and therefore named XKH1. When expressed by means of cRNA injection into frog oocytes, XKH1 solely exhibited at physiologic ionic strength hyaluronidase activity at neutral pH and in weakly acidic solutions. The enzyme was inactive below pH 5.4. In addition to hyaluronic acid hydrolysis, chondroitin sulfate also was degraded at low yield as assessed by fluorophore-assisted carbohydrate electrophoresis analysis of the degradation products. The enzyme is sorted to the outer surface of the cell membrane of XKH1 expressing oocytes. From there, it could not be removed by phospholipase C nor was secreted hyaluronidase activity detectable. We conclude that XKH1 represents a membrane-bound hyaluronan-degrading enzyme exclusively expressed in cells of the adult frog kidney where it either may be involved in the reorganization of the extracellular architecture or in supporting physiological demands for proper renal functions.