The interplay of increased urea synthesis and reduced ammonia production in the African lungfish Protopterus aethiopicus during 46 days of aestivation in a mucus cocoon

This study was undertaken to test the hypothesis that the rate of urea synthesis in Protopterus aethiopicus was up-regulated to detoxify ammonia during the initial phase of aestivation in air (day 1-day 12), and that a profound suppression of ammonia production occurred at a later phase of aestivation (day 35-day 46) which eliminated the need to sustain the increased rate of urea synthesis. Fasting apparently led to a greater rate of nitrogenous waste excretion in P. aethiopicus in water, which is an indication of increases in production of endogenous ammonia and urea probably as a result of increased proteolysis and amino acid catabolism for energy production. However, 46 days of fasting had no significant effects on the ammonia or urea contents in the muscle, liver, plasma and brain. In contrast, there were significant decreases in the muscle ammonia content in fish after 12, 34 or 46 days of aestivation in air when compared with fish fasting in water. Ammonia was apparently detoxified to urea because urea contents in the muscle, liver, plasma and brain of P. aethiopicus aestivated for 12, 34 or 46 days were significantly greater than the corresponding fasting control; the greatest increases in urea contents occurred during the initial 12 days. There were also significant increases in activities of some of the hepatic ornithine-urea cycle enzymes from fish aestivated for 12 or 46 days. Therefore, contrary to a previous report on P. aethiopicus, our results demonstrated an increase in the estimated rate of urea synthesis (2.8-fold greater than the day 0 fish) in this lungfish during the initial 12 days of aestivation. However, the estimated rate of urea synthesis decreased significantly during the next 34 days. Between day 35 and day 46 (12 days), urea synthesis apparently decreased to 42% of the day 0 control value, and this is the first report of such a phenomenon in African lungfish undergoing aestivation. On the other hand, the estimated rate of ammonia production in P. aethiopicus increased slightly (14.7%) during the initial 12 days of aestivation as compared with that in the day 0 fish. By contrast, the estimated rate of ammonia production decreased by 84% during the final 12 days of aestivation (day 35-day 46) compared with the day 0 value. Therefore, it can be concluded that P. aethiopicus depended mainly on increased urea synthesis to ameliorate ammonia toxicity during the initial phase of aestivation, but during prolonged aestivation, it suppressed ammonia production profoundly, eliminating the need to increase urea synthesis which is energy-intensive.     

Effects of hypoxia on the energy status and nitrogen metabolism of African lungfish during aestivation in a mucus cocoon

We examined the energy status, nitrogen metabolism and hepatic glutamate dehydrogenase activity in the African lungfish Protopterus annectens during aestivation in normoxia (air) or hypoxia (2% O(2) in N(2)), with tissues sampled on day 3 (aerial exposure with preparation for aestivation), day 6 (entering into aestivation) or day 12 (undergoing aestivation). There was no accumulation of ammonia in tissues of fish exposed to normoxia or hypoxia throughout the 12-day period. Ammonia toxicity was avoided by increased urea synthesis and/or decreased endogenous N production (as ammonia), but the dependency on these two mechanisms differed between the normoxic and the hypoxic fish. The rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the normoxic fish. By contrast, the rate of N production in the hypoxic fish decreased by 58%, with no increase in the rate of urea synthesis. Using in vivo (31)P NMR spectroscopy, it was demonstrated that hypoxia led to significantly lower ATP concentration on day 12 and significantly lower creatine phosphate concentration on days 1, 6, 9 and 12 in the anterior region of the fish as compared with normoxia. Additionally, the hypoxic fish had lower creatine phosphate concentration in the middle region than the normoxic fish on day 9. Hence, lowering the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing N production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase (GDH) amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, our results indicate for the first time that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia.     

Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, <Emphasis Type="Italic">Protopterus annectens</Emphasis>, during aestivation or exposure to ammonia

This study aimed to obtain the full sequence of carbamoyl phosphate synthetase III (cps III) from, and to determine the mRNA expression of cps III in, the liver of P. annectens during aestivation in air, hypoxia or mud, or exposure to environmental ammonia (100 mmol l⁻¹ NH₄Cl). The complete coding cDNA sequence of cps III from the liver of P. annectens consisted of 4530 bp, which coded for 1,510 amino acids with an estimated molecular mass of 166.1 kDa. The Cps III of P. annectens consisted of a mitochondrial targeting sequence of 44 amino acid residues, a GAT domain spanning from tyrosine 45 to isoleucine 414, and a methylglyoxal synthase-like domain spanning from valine 433 to arginine 1513. Two cysteine residues (cysteine 1337 and cysteine 1347) that are characteristic of N-acetylglutamate dependency were also present. The critical Cys-His-Glu catalytic triad (cysteine 301, histidine 385 and glutamate 387) together with methionine 302 and glutamine 305 affirmed that P. annectens expressed Cps III and not Cps I. A comparison of the translated amino acid sequence of Cps III from P. annectens with CPS sequences from other animals revealed that it shared the highest similarity with elasmobranch Cps III. A phylogenetic analysis indicates that P. annectens CPS III could have evolved from Cps III of elasmobranchs. Indeed, Cps III from P. annectens used mainly glutamine as the substrate, and its activity decreased significantly when glutamine and ammonia were included together in the assay system. There were significant increases (9- to 12-fold) in the mRNA expression of cps III in the liver of fish during the induction phase (days 3 and 6) of aestivation in air. Aestivation in hypoxia or in mud had a delayed effect on the increase in the mRNA expression of cps III, which extended beyond the induction phase of aestivation, reiterating the importance of differentiating effects that are intrinsic to aestivation from those intrinsic to hypoxia. Furthermore, results from this study confirmed that environmental ammonia exposure led to a significant increase in the mRNA expression of cps III in the liver of P. annectens, alluding to the important functional role of urea not only as a product of ammonia detoxification but also as a putative internal cue for aestivation.     

Changes in salinity and ionic compositions can act as environmental signals to induce a reduction in ammonia production in the African lungfish Protopterus dolloi

The slender African lungfish, Protopterus dolloi, does not aestivate in a subterranean mud cocoon, but is capable of aestivating inside a layer of dried mucus on land during drought. In this study, we aimed to elucidate if a slight increase in salinity in association with changes in the ionic composition could act as signals for P. dolloi to decrease endogenous ammonia production, in preparation for aestivation when the external medium dries up. Specimens of P. dolloi exposed to 3 per thousand water for 6 days exhibited consistently lower daily urea excretion rate than the freshwater control. This led to significant decreases in the cumulative total nitrogenous wastes excreted on days 3, 5 and 6. On day 6, there were decreases in urea contents in various tissues and organs. Taken together, these results suggest that there was a decrease in the rate of urea synthesis, the magnitude of which was greater than the decrease in the rate of urea excretion, and therefore resulted in decreases in internal urea contents. A decrease in the rate of urea synthesis should result in a decrease in the rate of glutamine utilization, and subsequently led to the accumulations of glutamine and/or ammonia. However, there were no changes in contents of glutamine and ammonia in various tissues and organs in the experimental animals. A logical explanation for this is that there must be a simultaneous reduction in ammonia production; if not, ammonia would accumulate due to the decrease in rate of urea synthesis. Since fish were unfed during the experiment, endogenous ammonia must be derived mainly from amino acid catabolism. Therefore, these results suggest that a suppression of amino acid catabolism occurred in specimens exposed to 3 per thousand for 6 days. The differences in effects of freshwater and 3 per thousand water on endogenous ammonia production could not be due to food deprivation because both groups of fish were fasted for the same period. Because control and experimental fish were kept in water and because there were no changes in the wet mass of the fish and blood osmolality before and after the experiment, dehydration did not occur. Furthermore, both groups of fish have comparable blood pH, pO2 and pCO2 on day 6 as they had free access to air, and therefore CO2 retention could be eliminated as the initiating factor of suppressed endogenous ammonia production. In conclusion, our results suggest that P. dolloi could respond to increases in salinity and changes in ionic composition in the external medium by suppressing ammonia production in preparation for aestivation when the water dries up.     

Differential Gene Expression in the Liver of the African Lungfish,Protopterus annectens,after 6 Months of Aestivation in Air or 1 Day of Arousal from 6 Months of Aestivation

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase) of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of aestivation in the liver of P. annectens.     

Effects of intra-peritoneal injection with NH4Cl, urea, or NH4Cl+urea on nitrogen excretion and metabolism in the African lungfish Protopterus dolloi

This study aimed to (1) determine if ammonia (as NH(4)Cl) injected intra-peritoneally into the ureogenic slender African lungfish, Protopterus dolloi, was excreted directly rather than being converted to urea; (2) examine if injected urea was retained in this lungfish, leading to decreases in liver arginine and brain tryptophan levels, as observed during aestivation on land; and (3) elucidate if increase in internal ammonia level would affect urea excretion, when ammonia and urea are injected simultaneously into the fish. Despite being ureogenic, P. dolloi rapidly excreted the excess ammonia as ammonia within the subsequent 12 h after NH(4)Cl was injected into its peritoneal cavity. Injected ammonia was not detoxified into urea through the ornithine-urea cycle, probably because it is energetically intensive to synthesize urea and because food was withheld before and during the experiment. In addition, injected ammonia was likely to stay in extracellular compartments available for direct excretion. At hour 24, only a small amount of ammonia accumulated in the muscle of these fish. In contrast, when urea was injected intra-peritoneally into P. dolloi, only a small percentage (34%) of it was excreted during the subsequent 24-h period. A significant increase in the rate of urea excretion was observed only after 16 h. At hour 24, significant quantities of urea were retained in various tissues of P. dolloi. Injection with urea led to an apparent reduction in endogenous ammonia production, a significant decrease in the hepatic arginine content, and a significantly lower level of brain tryptophan in this lungfish. All three phenomena had been observed previously in aestivating P. dolloi. Hence, it is logical to deduce that urea synthesis and accumulation could be one of the essential factors in initiating and perpetuating aestivation in this lungfish. Through the injection of NH(4)Cl + urea, it was demonstrated that an increase in urea excretion occurred in P. dolloi within the first 12 h post-injection, which was much earlier than that of fish injected with urea alone. These results suggest that urea excretion in P. dolloi is likely to be regulated by the level of internal ammonia in its body.     

Nitrogen metabolism and excretion in the swamp eel, Monopterus albus, during 6 or 40 days of estivation in mud

Monopterus albus inhabits muddy ponds, swamps, canals, and rice fields, where it can burrow into the moist earth, and it survives for long periods during the dry summer season. However, it had been reported previously that mortality increased when M. albus was exposed to air for 8 d or more. Thus, the objective of this study was to elucidate the strategies adopted by M. albus to defend against ammonia toxicity during 6 or 40 d of estivation in mud and to evaluate whether these strategies were different from those adopted by fish to survive 6 d of aerial exposure. Ammonia and glutamine accumulations occurred in the muscle and liver of fish exposed to air (normoxia) for 6 d, indicating that ammonia was detoxified to glutamine under such conditions. In contrast, ammonia accumulation occurred only in the muscle, with no increases in glutamine or glutamate contents in all tissues, of fish estivated in mud for 6 d. Similar results were obtained from fish estivated in mud for 40 d. While estivating in mud prevented excessive water loss through evaporation, M. albus was exposed to hypoxia, as indicated by significant decreases in blood P(O(2)), muscle energy charge, and ATP content in fish estivated in mud for 6 d. Glutamine synthesis is energy intensive, and that could be the reason why M. albus did not depend on glutamine synthesis to defend against ammonia toxicity when a decrease in ATP supply occurred. Instead, suppression of endogenous ammonia production was adopted as the major strategy to ameliorate ammonia toxicity when M. albus estivated in mud. Our results suggest that a decrease in O(2) level in the mud could be a more effective signal than an increase in internal ammonia level during aerial exposure to induce a suppression of ammonia production in M. albus. This might explain why M. albus is able to estivate in mud for long periods (40 d) but can survive in air for only <10 d.     

Increases in urea synthesis and the ornithine-urea cycle capacity in the giant African snail, Achatina fulica, during fasting or aestivation, or after the injection with ammonium chloride

The objectives of this study are to determine whether a full complement of ornithine-urea cycle (OUC) enzymes is present in the hepatopancreas of the giant African snail Achatina fulica, and to investigate whether the rate of urea synthesis and the OUC capacity can be up-regulated during 23 days of fasting or aestivation, or 24 hr post-injection with NH(4)Cl (10 micromol g(-1) snail) into the foot muscle. A. fulica is ureotelic and a full complement of OUC enzymes, including carbamoyl phosphate synthetase III (CPS III), was detected from its hepatopancreas. There were significant increases in the excretion of NH(4)(+), NH(3) and urea in fasting A. fulica. Fasting had no significant effect on the tissue ammonia contents, but led to a progressive accumulation of urea, which was associated with an 18-fold increase in the rate of urea synthesis. Because fasting took place in the presence of water and because there was no change in water contents in the foot muscle and hepatopancreas, it can be concluded that the function of urea accumulation in fasting A. fulica was unrelated to water retention. Aestivation in arid conditions led to a non-progressive accumulation of urea in A. fulica. During the first 4 days and the last 3 days of the 23-day aestivation period, experimental snails exhibited significantly greater rates of urea synthesis compared with fasted snails. These increases were associated with significant increases in activities of various OUC enzymes, except CPS III, in the hepatopancreas. However, the overall urea accumulation in snails aestivated and snails fasted for 23 days were comparable. Therefore, the classical hypothesis that urea accumulation occurred to prevent water loss through evaporation during aestivation in terrestrial pulmonates may not be valid. Surprisingly, there were no accumulations of ammonia in the foot muscle and hepatopancreas of A. fulica 12 or 24 hr after NH(4)Cl was injected into the foot muscle. In contrast, the urea content in the foot muscle of A. fulica increased 4.5- and 33-fold at hour 12 and hour 24, respectively, and the respective increases in the hepatopancreas were 4.9- and 32-fold. The exogenous ammonia injected into A. fulica was apparently detoxified completely to urea. The urea synthesis rate increased 148-fold within the 24-hr experimental period, which could be the greatest increase known among animals. Simultaneously, there were significant increases in activities of glutamine synthetase (2.5-fold), CPS III (3.1-fold), ornithine transcarbamoylase (2.3-fold), argininosuccinate synthetase+lyase (13.6-fold) and arginase (3.5-fold) in the hepatopancreas 12 hr after the injection of NH(4)Cl. Taken altogether, our results support the view that the primary function of urea synthesis through the OUC in A. fulica is to defend against ammonia toxicity, but suggest that urea may have more than an excretory role in terrestrial pulmonates capable of aestivation.     

Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens,and their mRNA expression levels in the liver,kidney, brain and skeletal muscle during aestivation

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia–reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.     

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during Pregnancy Induced Hypertension

Changes in [³⁵S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails,Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest thatO. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.Abbreviations dpm radioactive disintegrations per minute - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - SRP stress related protein     

Energy loss in an aestivating population of the tropical snail pila globosa

The dried peripheral area of pond Idumban (62 ha) increased from 3.2 ha in January to 3 1.9 ha in April. Pila globosa, which were abundant in the littoral area, did not commence aestivation during this period, perhaps due to low temperature and/or high dissolved oxygen content. The number of aestivating snails averaged 0.5/m² in May, 1973 (3.6% of the total population) and it increased to 1.1/M² in September (26.2%). Biomass of the snail increased from 3.5 to 19.9 g dry weight (including shell)/M². Number of aestivating snails increased from 0.4/m² (5.2% of the total population) in May 1974 to 0.8/m² (11.1%) in July and the biomass from 4.1 g/m² to 10.7 g/m². Availability of dried area for aestivation increased from 5.3 to 23.7 ha in 1973 and from 13.5 to 30.2 ha in 1974.Monthly observations made on the marked snails forced to aestivate at 7.5, 15.0, 22.5 and 30.0 cm depth in the pond during May, revealed that temperature above 35°C and moisture below 5% were critical. Mortality and weight loss decreased in the snails forced to aestivate at increasing depth. Random observations indicated that 83% of the aestivating snails buried themselves at 15 cm depth in the pond. On the whole, 98,480 snails (592 Kg) and 115,270 (758 Kg) died during aestivation in 1973 and 1974 respectively. Of the total weight loss, the energy lost via metabolism contributed only a small fraction of 2.2% (12 Kg) and 2.1% (15 Kg) during these years. Considering the total aestivation area, the snails which succumbed averaged only 0.4/m²/year (2.5 g/m²/year). On an average, dry substance equivalent to about 2.6 mg dry weight/ g dry weight of snail/ day (3.7 gcal/ g live snail/ day) was lost on metabolism by the aestivating snails, i.e. the metabolic level of the aestivating snail was about 1 / 18th of that of the actively feeding snail.     

Differential gene expression in the liver of the African lungfish, <Emphasis Type="Italic">Protopterus annectens</Emphasis>, after 6 days of estivation in air

This study aimed to identify estivation-specific gene clusters through the determination of differential gene expressions in the liver of Protopterus annectens after 6 days of estivation in a mucus cocoon in air (normoxia) using suppression subtractive hybridization polymerase chain reaction. Our results demonstrated that 6 days of estivation in normoxia led to up-regulation of mRNA expressions of several genes related to urea synthesis, including carbamoyl phosphate synthetase (Cps), argininosuccinate synthetase and glutamine synthetase. They indicate that increased urea synthesis, despite being energy-intensive, is an important adaptive response of estivation. They also offer indirect support to the proposition that urea synthesis in this lungfish involved a Cps that uses glutamine as a substrate. In addition, up- or down-regulation of several gene clusters occurred in the liver of P. annectens after 6 days of estivation in normoxia. These estivation-specific genes were involved in the prevention of clot formation, activation of the lectin pathway for complement activation, conservation of minerals (e.g. iron and copper) and increased production of hemoglobin beta. Since there were up- and down-regulation of mRNA expressions of genes related to ribosomal proteins and translational elongation factors, there could be simultaneous increases in protein degradation and protein synthesis during the first 6 days (the induction phase) of estivation, confirming the importance of reconstruction of protein structures in preparation for the maintenance phase of estivation.     

Postprandial increases in nitrogenous excretion and urea synthesis in the Chinese soft-shelled turtle, <Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, with a special emphasis on the role of urea synthesis in ammonia detoxification. P. sinensis is ureogenic and possesses a full complement of ornithine-urea cycle enzymes in its liver. It is primarily ureotelic in water, and the estimated rate of urea synthesis in unfed animals was equivalent to only 1.5% of the maximal capacity of carbamoyl phosphate synthetase I (CPS I) in its liver. Approximately 72 h was required for P. sinensis to completely digest a meal of prawn meat. During this period, there were significant increases in ammonia contents in the stomach at hour 24 and in the intestine between hours 12 and 36, which could be a result of bacterial activities in the intestinal tract. However, ammonia contents in the liver, muscle, brain and plasma remained unchanged throughout the 72-h post-feeding. In contrast, at hour 24, urea contents in the stomach, intestine, liver, muscle, brain and plasma increased significantly by 2.9−, 3.5−, 2.6−, 2.9−, 3.4 and 3.0-fold, respectively. In addition, there was a 3.3- to 8.0−fold increase in the urea excretion rate between hours 0 and 36 post-feeding, which preceded the increase in ammonia excretion between hours 12 and 48. By hour 48, 68% of the assimilated N from the feed was excreted, 54% of which was excreted as urea-N. The rate of urea synthesis apparently increased sevenfold during the initial 24 h after feeding, which demanded only 10% of the maximal CPS I capacity in P. sinensis. The postprandial detoxification of ammonia to urea in P. sinensis effectively prevented postprandial surges in ammonia contents in the plasma and other tissues, as observed in other animals, during the 72-h period post-feeding. In addition, postprandial ammonia toxicity was ameliorated by increased transamination and synthesis of certain amino acids in the liver and muscle of P. sinensis. After feeding, a slight but significant increase in the glutamine content occurred in the brain at hour 24, indicating that the brain might experience a transient increase in ammonia and ammonia was detoxified to glutamine.     

The NOS/NO system in an example of extreme adaptation: The African lungfish

African dipnoi (lungfish) are aestivating fish and obligate air breathers that, throughout their complex life cycle, undergo remarkable morpho-functional organ readjustment from biochemical to morphological level. In the present review we summarize the changes of the NOS/NO (Nitric Oxide Synthase/Nitric Oxide) system occurring in lungs, gills, kidney, heart, and myotomal muscle of African lungfish of the genus Protopterus (P. dolloi and P. annectens), in relation to the switch from freshwater to aestivation, and vice-versa. In particular, the expression and localization patterns of NOS, and its protein partners Akt, Hsp-90 and HIF-1α, have been discussed, together with the apoptosis rate, evaluated by TUNEL technique.We hypothesize that all these molecular components are crucial in signalling transduction/integration networks induced by environmental challenges (temperature, dehydration, inactivity)experienced at the beginning, during, and at the end of the dry season.     

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     

Excretory nitrogen metabolism in the Chinese fire-belly newt<Emphasis Type="Italic"> Cynops orientalis</Emphasis> in water,on land,or in high concentrations of environmental ammonia

The Chinese fire-belly newt Cynops orientalis reverts to an aquatic mode of living when sexually mature. Despite living in water, sexually mature C. orientalis maintained high capacity for hepatic urea synthesis. However, it had a lower rate of urea production than other terrestrial amphibians because endogenous ammonia could diffuse out to the external medium as NH₃. This conserves cellular energy because urea synthesis is energetically expensive. Simultaneously, C. orientalis also reduced the rate of urea excretion, and excreted 33% of the total nitrogenous waste as ammonia. Upon exposure to land, C. orientalis increased the rate of urea synthesis from accumulating endogenous ammonia. The increased rate of urea synthesis was within the inherent capacity of the hepatic ornithine–urea cycle; there was no induction of hepatic carbamoyl phosphate synthetase or ornithine transcarbamoylase activities and there was no reduction in ammonia production. When exposed to water containing 75 mmol.l^–1 NH₄Cl, the rates of both urea synthesis and urea excretion increased. Under such experimental conditions, the ornithine–urea cycle may be operating close to its limit; glutamine began to accumulate in the body, and endogenous ammonia production via amino acid catabolism was reduced.Abbreviations CPS carbamoyl phosphate synthetase - FAA free amino acid - OTC ornithine transcarbamoylase - OUC ornithine–urea cycle - TCA trichloroacetic acid Communicated by I.D. Hume     

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.     

Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

Air breathing and ammonia excretion in the giant mudskipper, Periophthalmodon schlosseri

The giant mudskipper, Periophthalmodon schlosseri, is an amphibious, obligate, air-breathing teleost fish. It uses its buccal cavity for air breathing and for taking and holding large gulps of air. These fish live in mud burrows at the top of the intertidal zone of mangrove mudflats; the burrow water may be hypoxic and hypercapnic and have high ammonia levels. The buccal epithelium is highly vascularized, with small diffusion distances between air and blood. The gill epithelium is densely packed with mitochondria-rich cells. Periophthalmodon schlosseri can maintain tissue ammonia levels in the face of high ammonia concentrations in the water. This is probably achieved by active ammonium ion transport across the mitochondria-rich cells via an apical Na/H+(NH4+) exchanger and a basolateral Na/K+(NH4+) ATPase. When exposed to air, the animal reduces ammonia production, but there is some increase in tissue ammonia levels after 24 h. There is no detoxification by increased production of glutamine or urea, but there is partial amino acid catabolism, leading to the accumulation of alanine. CO2 production and proton excretion cause acidification of the burrow water to reduce ammonia toxicity. The skin has high levels of cholesterol and saturated fatty acids decreasing membrane fluidity and gas, and therefore ammonia, permeability. Exposure to elevated environmental ammonia further decreases membrane permeability. Acidification of the environment and having a skin with a low NH3 permeability reduces ammonia influx, so that the fish can maintain tissue ammonia levels by active ammonium ion excretion, even in water containing high levels of ammonia.