The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.

Control of breathing in African lungfish (Protopterus dolloi): a comparison of aquatic and cocooned (terrestrialized) animals.

African lungfish, Protopterus dolloi exhibited constant rates of O(2) consumption before (0.95+/-0.07 mmol kg(-1) h(-1)), during (1.21+/-0.32 mmol kg(-1) h(-1)) and after (1.14+/-0.14 mmol kg(-1) h(-1)) extended periods (1-2 months) of terrestrialization while cocooned. Although a breathing event in terrestrialized fish consisted of multiple bouts of inspiration and expiration in rapid succession, the mean frequency of pulmonary breathing events was unaltered in the terrestrialized fish (16.7+/-1.4 h(-1)versus 20.1+/-4.9 h(-1) in the aquatic and terrestrialized fish, respectively). Hypoxia (approximately 20 mmHg) increased the frequency of breathing events by 16 and 23 h(-1) in the aquatic and terrestrialized fish, respectively. Hyperoxia (approximately 550 mmHg) decreased breathing event frequency by 10 and 15 h(-1) in the aquatic and terrestrialized animals. Aquatic hypercapnia (approximately 37.5 mmHg) increased pulmonary breathing frequency (from 15.3+/-2.3 to 28.7+/-5.4 h(-1)) in free swimming lungfish, whereas aerial hypercapnia was without effect in aquatic or terrestrialized fish.

Defense against environmental ammonia toxicity in the African lungfish, Protopterus aethiopicus: Bimodal breathing, skin ammonia permeability and urea synthesis.

This study aimed to determine how the African lungfish Protopterus aethiopicus defended against ammonia toxicity when confronted with high concentrations (30 or 100 mmoll(-1)) of environmental ammonia. Exposure to 100 mmoll(-1) of NH(4)Cl for 1 or 6 days had no significant effect on the rate of O(2) uptake from water or from air, and the rate of total O(2) consumption. Using an Ussing-like apparatus, we report for the first time that the skin of P. aethiopicus had low permeability (1.26 x 10(-4) micromol min(-1)cm(-1)) to NH(3)in vitro. Indeed, the influx of exogenous ammonia into fish exposed to 30 mmoll(-1) NH(4)Cl was low (0.117 micromol min(-1) 100g(-1) fish). As a result, P. aethiopicus could afford to maintain relatively low ammonia contents in plasma, muscle, liver and brain even after 6 days of exposure to 100 mmoll(-1) NH(4)Cl. Surprisingly, fish exposed to 30 or 100 mmoll(-1) NH(4)Cl had comparable ammonia contents in the muscle and the brain in spite of the big difference (70 mmoll(-1)) in environmental ammonia concentrations. Significant increases in urea contents occurred in various tissues of fish exposed to 30 mmoll(-1) NH(4)Cl for 6 days, but there were no significant differences in tissue urea contents between fish exposed to 30 mmoll(-1) and 100 mmoll(-1) NH(4)Cl. Between days 3 and 6, the rate of urea excretion in fish exposed to 30 mmoll(-1) NH(4)Cl was significantly greater than that of the control. By contrast, there was no significant difference in urea excretion rates between fish exposed to 100 mmoll(-1) NH(4)Cl and control fish throughout the 6-day period, and such a phenomenon has not been reported before for other lungfish species. Thus, our results suggest that P. aethiopicus was capable of decreasing the NH(3) permeability of its body surface when exposed to high concentrations of environmental ammonia. Indeed, after 6 days of exposure to 100 mmoll(-1) NH(4)Cl, the NH(3) permeability constant of the skin (0.55 x 10(-4) micromol min(-1)cm(-1)) decreased to half of that of the control. A decrease in the already low cutaneous NH(3) permeability and an increased urea synthesis, working in combination, allowed P. aethiopicus to effectively defend against environmental ammonia toxicity without elevating the plasma ammonia level. Therefore, unlike other fishes, glutamine and alanine contents did not increase in the muscle and liver, and there was no accumulation of glutamine in the brain, even when the fish was immersed in water containing 100 mmoll(-1) NH(4)Cl.

Differential gene expression in the liver of the African lungfish, Protopterus annectens, 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.

Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, 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) NH(4)Cl). 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.

Roles of intestinal glutamate dehydrogenase and glutamine synthetase in environmental ammonia detoxification in the euryhaline four-eyed sleeper, Bostrychus sinensis.

This study aimed to examine the hypothesis that intestinal glutamate dehydrogenase (GDH) and glutamine synthetase (GS) could be involved in ammonia detoxification in the euryhaline Bostrychus sinensis exposed to ammonia in a hyperosmotic environment, whereby drinking was essential for osmoregulation. Our results indicate that there was a significant increase in ammonia content in the intestine of B. sinensis exposed to 15 mmol l(-1) NH(4)Cl in seawater (pH 7.0) for 6 days. There were also significant increases in the amination and deamination activities and protein abundance of intestinal GDH. The GDH amination/deamination ratio remained unchanged, indicating that there could be increases in the turnover of glutamate. However, the difference between the amination and deamination activities increased 2-fold, implying that there could be an increase in glutamate formation in the intestine. Since the intestinal glutamate content remained unchanged, excess glutamate formed might have been channeled into other amino acids and/or transported to other organs. Indeed, the intestinal glutamine content increased significantly by 2-fold, with a significant increase in the activity and protein abundance of intestinal GS. Since the magnitude of glutamine accumulation in the intestine was lower than those in liver and muscle, which lacked changes in GDH activities, intestinal glutamate could have been shuttled to liver and muscle to facilitate increased synthesis of glutamine therein. By contrast, when fish were exposed to a much higher concentration (30 mmol l(-1)) of NH(4)Cl in 5 per thousand water (pH. 7.0), the magnitude of increase in ammonia content in the intestine was less prominent, and there were no changes in activities and kinetic properties of intestinal GDH. Therefore, it can be concluded that the intestine of B. sinensis was involved in the defense against ammonia toxicity during exposure to ammonia in a hyperosmotic medium.

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.