Signal molecule changes in the gills and lungs of the African lungfish Protopterus annectens, during the maintenance and arousal phases of aestivation.

African lungfishes are obligate air breathers, with reduced gills and pulmonary breathing throughout their life. During the dry season they aestivate on land, with the collapse of secondary lamellae of their gills and the establishment of an exclusive aerial ventilation through the vascularization and expansion of their lungs. To date, the mechanisms underlining the respiratory organ remodeling in aestivating lungfishes are unknown. This study aimed to identify key switch components of the stress-induced signal transduction networks implicated in both rapid and medium-long term remodeling of the gills and lungs of the African lungfish Protopterus annectens during aestivation. Through immunofluorescence microscopy and Western blotting, the localization and the expression of nitric oxide synthase (NOS), Akt, Hsp-90 and HIF-1α were evaluated in both gills and lungs exposed to three experimental conditions: freshwater (FW), 6 months of experimentally induced aestivation (6mAe), and 6 days after arousal from 6 months of aestivation (6mAe6d). After 6mAe, the expression of NOS (p-eNOS antibody), Akt (p-Akt antibody), and Hsp-90 decreased in the gills, while NOS and Hsp-90 expression increased with Akt remained unchanged in the lungs. Upon 6mAe6d, NOS, Akt and Hsp-90 expression in the gills returned to the respective FW values. In the lungs of the aroused fish, NOS and Akt decreased to their respective FW levels, while Hsp-90 expression was enhanced with respect to aestivation. In both respiratory organs, the qualitative and quantitative patterns of HIF-1α expression correlated inversely to those of NOS. Overall, our findings suggest that the molecular components of the NOS/NO system changed in a tissue-specific manner in parallel with organ readjustment in the gills and lungs of P. annectens during aestivation and arousal.

Excretory nitrogen metabolism and defence against ammonia toxicity in air-breathing fishes.

With the development of air-breathing capabilities, some fishes can emerge from water, make excursions onto land or even burrow into mud during droughts. Air-breathing fishes have modified gill morphology and morphometry and accessory breathing organs, which would tend to reduce branchial ammonia excretion. As ammonia is toxic, air-breathing fishes, especially amphibious ones, are equipped with various strategies to ameliorate ammonia toxicity during emersion or ammonia exposure. These strategies can be categorized into (1) enhancement of ammonia excretion and reduction of ammonia entry, (2) conversion of ammonia to a less toxic product for accumulation and subsequent excretion, (3) reduction of ammonia production and avoidance of ammonia accumulation and (4) tolerance of ammonia at cellular and tissue levels. Active ammonia excretion, operating in conjunction with lowering of ambient pH and reduction in branchial and cutaneous NH3 permeability, is theoretically the most effective strategy to maintain low internal ammonia concentrations. NH3 volatilization involves the alkalization of certain epithelial surfaces and requires mechanisms to prevent NH3 back flux. Urea synthesis is an energy-intensive process and hence uncommon among air-breathing teleosts. Aestivating African lungfishes detoxify ammonia to urea and the accumulated urea is excreted following arousal. Reduction in ammonia production is achieved in some air-breathing fishes through suppression of amino acid catabolism and proteolysis, or through partial amino acid catabolism leading to alanine formation. Others can slow down ammonia accumulation through increased glutamine synthesis in the liver and muscle. Yet, some others develop high tolerance of ammonia at cellular and tissue levels, including tissues in the brain. In summary, the responses of air-breathing fishes to ameliorate ammonia toxicity are many and varied, determined by the behaviour of the species and the nature of the environment in which it lives.

Nitric oxide synthase-dependent "on/off" switch and apoptosis in freshwater and aestivating lungfish, Protopterus annectens: skeletal muscle versus cardiac muscle.

African lungfishes (Protopterus spp.) are obligate air breathers which enter in a prolonged torpor (aestivation) in association with metabolic depression, and biochemical and morpho-functional readjustments during the dry season. During aestivation, the lungfish heart continues to pump, while the skeletal muscle stops to function but can immediately contract during arousal. Currently, nothing is known regarding the orchestration of the multilevel rearrangements occurring in myotomal and myocardial muscles during aestivation and arousal. Because of its universal role in cardio-circulatory and muscle homeostasis, nitric oxide (NO) could be involved in coordinating these stress-induced adaptations. Western blotting and immunofluorescence microscopy on cardiac and skeletal muscles of Protopterus annectens (freshwater, 6months of aestivation and 6days after arousal) showed that expression, localization and activity of the endothelial-like nitric oxide synthase (eNOS) isoform and its partners Akt and Hsp-90 are tissue-specifically modulated. During aestivation, phospho-eNOS/eNOS and phospho-Akt/Akt ratios increased in the heart but decreased in the skeletal muscle. By contrast, Hsp-90 increased in both muscle types during aestivation. TUNEL assay revealed that increased apoptosis occurred in the skeletal muscle of aestivating lungfish, but the myocardial apoptotic rate of the aestivating lungfish remained unchanged as compared with the freshwater control. Consistent with the preserved cardiac activity during aestivation, the expression of apoptosis repressor (ARC) also remained unchanged in the heart of aestivating and aroused fish as compared with the freshwater control. Contrarily, ARC expression was strongly reduced in the skeletal muscle of aestivating lungfish. On the whole, our data indicate that changes in the eNOS/NO system and cell turnover are implicated in the morpho-functional readjustments occurring in lungfish cardiac and skeletal muscle during the switch from freshwater to aestivation, and between the maintenance and arousal phases of aestivation.

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.

Cytochrome c oxidase is regulated by modulations in protein expression and mitochondrial membrane phospholipid composition in estivating African lungfish.

We examined some of the potential mechanisms lungfish (Protopterus dolloi) use to regulate cytochrome c oxidase (CCO), during metabolic depression. CCO activity was reduced by 67% in isolated liver mitochondria of estivating fish. This was likely accomplished, in part, by the 46% reduction in CCO subunit I protein expression in the liver. No change in the mRNA expression levels of CCO subunits I, II, III, and IV were found in the liver, suggesting CCO is under translational regulation; however, in the kidney, messenger limitation may be a factor as the expression of subunits I and II were depressed ( approximately 10-fold) during estivation, suggesting tissue-specific mechanisms of regulation. CCO is influenced by mitochondrial membrane phospholipids, particularly cardiolipin (CL). In P. dolloi, the phospholipid composition of the liver mitochondrial membrane changed during estivation, with a approximately 2.3-fold reduction in the amount of CL. Significant positive correlations were found between CCO activity and the amount of CL and phosphatidylethanolamine within the mitochondrial membrane. It appears CCO activity is regulated through multiple mechanisms in P. dolloi, and individual subunits of CCO are regulated independently, and in a tissue-specific manner. It is proposed that altering the amount of CL within the mitochondrial membrane may be a means of regulating CCO activity during metabolical depression in the African lungfish, P. dolloi.

Nitrogen metabolism and excretion during aestivation.

In this chapter, up-to-date information on nitrogen metabolism and excretion in various aestivators is presented. Although aestivation involves long-term fasting and corporal torpor, adaptive responses with regard to excretory nitrogen metabolism exhibited by aestivators during aestivation differ from those exhibited by nonaestivators undergoing fasting or immobilization. Special efforts were made to address current issues pertaining to excretory nitrogen metabolism and related phenomena in aestivators. Adaptations exhibited by aestivators were discussed in relation to the induction, maintenance, and arousal phases of aestivation. For the induction phase, we included topics like urea as an internal induction signal for aestivation, alteration in the permeability of the skin to ammonia, and changes in rate of ammonia production and urea synthesis. For the maintenance phase, the emphasis was on protein synthesis and degradation, ammonia production, and urea synthesis and accumulation. For the arousal phase, the focus was on rehydration, urea excretion, and phenomena related to feeding. Adaptations exhibited by aestivators specifically to each of these three phases of aestivation are essential to the understanding of the overall aestivation process, but, at present, only limited information is available on excretory nitrogen metabolism in animals during the induction or arousal phases of aestivation. Therefore, future efforts should be made to identify adaptive responses particular to each of the three phases of aestivation in various aestivators.

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus.

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na(+)/K(+)-ATPase, H(+)-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC(50) value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na(+)/NH(4)(+)-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H(+)-ATPase and Na(+)/K(+)-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H(+)-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H(+)-ATPase and Rhcg1 to the similar non-Na(+)/K(+)-ATPase immunoreactive cell type would support a role for H(+)-ATPase in ammonia excretion via Rhcg by NH(4)(+) trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H(+)-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H(+)-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH(3) permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.

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.

Ionoregulatory physiology of two species of African lungfishes Protopterus dolloi and Protopterus annectens.

Basic ionoregulatory physiology was characterized in two species of African lungfish, slender African lungfish Protopterus dolloi and West African lungfish Protopterus annectens, largely under aquatic conditions. There were no substantive differences between the two species. Plasma [Na], [Cl] and [Ca] were only 60-80% of those typical of freshwater teleosts, and plasma Ca activity was particularly low. Unidirectional Na and Cl influx rates from water were also very low, only c. 10% of teleost values, whereas unidirectional Ca influx rates were comparable with teleost rates. Protopterus spp. were fed a 3% ration of bloodworms every 48 h. The bloodworm diet provided similar amounts of Na and Ca as uptake from water, but almost no Cl. Efflux rates of Na and Cl through the urine were greater than via the faeces, whereas the opposite was true for Ca. Net ion flux measurements and ionic balance sheet calculations indicated that (1) both water and dietary uptake routes are important for Na and Ca acquisition; (2) the waterborne route predominates for Cl uptake; (3) unidirectional ion effluxes across the body surface (gills and skin) rather than urine and faeces are the major routes of loss for Na, Cl and Ca. Tissues (muscle, liver, lung, kidney, intestine and heart) and plasma ions were also examined in P. dolloi'terrestrialized' in air for up to 5 months, during which plasma ion concentrations (Na, Cl, Ca and Mg) did not change and there were only a few alterations in tissue ions, that is, increased [Na] in intestine, decreased [Cl] in kidney and increased [Ca] in liver and kidney.

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.

Mechanisms of and defense against acute ammonia toxicity in the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis.

The objective of this study was to elucidate the mechanisms of acute ammonia toxicity in the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, and to examine how this turtle defended against a sublethal dose of NH(4)Cl injected into its peritoneal cavity. The ammonia and glutamine contents in the brains of turtles that succumbed within 3h to an intraperitoneal injection with a lethal dose (12.5 micromolg(-1) turtle) of NH(4)Cl were 21 and 4.4 micromolg(-1), respectively. Since the brain glutamine content increased to 8 micromolg(-1) at hour 6 and recovered thereafter in turtles injected with a sub-lethal dose of NH(4)Cl (7.5 micromolg(-1) turtle), it can be concluded that increased glutamine synthesis and accumulation was not the major cause of acute ammonia toxicity in P. sinensis. Indeed, the administration of l-methionine S-sulfoximine (MSO; 82 microgg(-1) turtle), a glutamine synthetase (GS) inhibitor, prior to the injection of a lethal dose of NH(4)Cl had no significant effect on the mortality rate. Although the prior administration of MSO led to an extension of the time to death, it was apparently a result of its effects on glutamate dehydrogenase and glutamate formation, instead of glutamine synthesis and accumulation, in the brain. By contrast, a prior injection with MK801 (1.6 microgg(-1) turtle), a NMDA receptor antagonist, reduced the 24h mortality of turtles injected with a lethal dose of NH(4)Cl by 50%. Thus, acute ammonia toxicity in P. sinensis was probably a result of glutamate dysfunction and the activation of NMDA receptors. NMDA receptor activation could also be exacerbated through membrane depolarization caused by the extraordinarily high level of ammonia (21 micromolg(-1) brain) in the brain of turtles that succumbed to a lethal dose of NH(4)Cl. One hour after the injection with a sub-lethal dose of NH(4)Cl, the brain of P. sinensis exhibited an extraordinarily high tolerance of ammonia (16 micromolg(-1) brain). The transient nature of ammonia accumulation indicates that P. sinensis could ameliorate ammonia toxicity through the suppression of endogenous ammonia production and/or the excretion of exogenous ammonia. Despite being ureogenic and ureotelic, only a small fraction of the exogenous ammonia was detoxified to urea. A major portion of ammonia was excreted unchanged, resulting in an apparent ammonotely in the experimental turtles. Since there were increases in total essential free amino acid contents in the brain, liver and muscle, it can be deduced that a suppression of amino acid catabolism had occurred, reducing the production of endogenous ammonia and hence alleviating the possibility of ammonia intoxication.

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.

Mechanisms of acid-base regulation in the African lungfish Protopterus annectens.

African lungfish Protopterus annectens utilized both respiratory and metabolic compensation to restore arterial pH to control levels following the imposition of a metabolic acidosis or alkalosis. Acid infusion (3 mmol kg(-1) NH(4)Cl) to lower arterial pH by 0.24 units increased both pulmonary (by 1.8-fold) and branchial (by 1.7-fold) ventilation frequencies significantly, contributing to 4.8-fold and 1.9-fold increases in, respectively, aerial and aquatic CO(2) excretion. This respiratory compensation appeared to be the main mechanism behind the restoration of arterial pH, because even though net acid excretion (J(net)H(+)) increased following acid infusion in 7 of 11 fish, the mean increase in net acid excretion, 184.5+/-118.5 micromol H(+) kg(-1) h(-1) (mean +/- s.e.m., N=11), was not significantly different from zero. Base infusion (3 mmol kg(-1) NaHCO(3)) to increase arterial pH by 0.29 units halved branchial ventilation frequency, although pulmonary ventilation frequency was unaffected. Correspondingly, aquatic CO(2) excretion also fell significantly (by 3.7-fold) while aerial CO(2) excretion was unaffected. Metabolic compensation consisting of negative net acid excretion (net base excretion) accompanied this respiratory compensation, with J(net)H(+) decreasing from 88.5+/-75.6 to -337.9+/-199.4 micromol H(+) kg(-1) h(-1) (N=8). Partitioning of net acid excretion into renal and extra-renal (assumed to be branchial and/or cutaneous) components revealed that under control conditions, net acid excretion occurred primarily by extra-renal routes. Finally, several genes that are involved in the exchange of acid-base equivalents between the animal and its environment (carbonic anhydrase, V-type H(+)-ATPase and Na(+)/HCO (-)(3) cotransporter) were cloned, and their branchial and renal mRNA expressions were examined prior to and following acid or base infusion. In no case was mRNA expression significantly altered by metabolic acid-base disturbance. These findings suggest that lungfish, like tetrapods, alter ventilation to compensate for metabolic acid-base disturbances, a mechanism that is not employed by water-breathing fish. Like fish and amphibians, however, extra-renal routes play a key role in metabolic compensation.

Metabolic organization of freshwater, euryhaline, and marine elasmobranchs: implications for the evolution of energy metabolism in sharks and rays.

To test the hypothesis that the preference for ketone bodies rather than lipids as oxidative fuel in elasmobranchs evolved in response to the appearance of urea-based osmoregulation, we measured total non-esterified fatty acids (NEFA) in plasma as well as maximal activities of enzymes of intermediary metabolism in tissues from marine and freshwater elasmobranchs, including: the river stingray Potamotrygon motoro (<1 mmol l(-1) plasma urea); the marine stingray Taeniura lymma, and the marine shark Chiloscyllium punctatum (>300 mmol l(-1) plasma urea); and the euryhaline freshwater stingray Himantura signifer, which possesses intermediate levels of urea. H. signifer also were acclimated to half-strength seawater (15 per thousand) for 2 weeks to ascertain the metabolic effects of the higher urea level that results from salinity acclimation. Our results do not support the urea hypothesis. Enzyme activities and plasma NEFA in salinity-challenged H. signifer were largely unchanged from the freshwater controls, and the freshwater elasmobranchs did not show an enhanced capacity for extrahepatic lipid oxidation relative to the marine species. Importantly, and contrary to previous studies, extrahepatic lipid oxidation does occur in elasmobranchs, based on high carnitine palmitoyl transferase (CPT) activities in kidney and rectal gland. Heart CPT in the stingrays was detectable but low, indicating some capacity for lipid oxidation. CPT was undetectable in red muscle, and almost undetectable in heart, from C. punctatum as well as in white muscle from T. lymma. We propose a revised model of tissue-specific lipid oxidation in elasmobranchs, with high levels in liver, kidney and rectal gland, low or undetectable levels in heart, and none in red or white muscle. Plasma NEFA levels were low in all species, as previously noted in elasmobranchs. D-beta-hydroxybutyrate dehydrogenase (d-beta-HBDH) was high in most tissues confirming the importance of ketone bodies in elasmobranchs. However, very low d-beta-HBDH in kidney from T. lymma indicates that interspecific variability in ketone body utilization occurs. A negative relationship was observed across species between liver glutamate dehydrogenase activity and tissue or plasma urea levels, suggesting that glutamate is preferentially deaminated in freshwater elasmobranchs because it does not need to be shunted to urea production as in marine elasmobranchs.

An investigation of the role of carbonic anhydrase in aquatic and aerial gas transfer in the African lungfish Protopterus dolloi.

Experiments were performed on bimodally breathing African lungfish Protopterus dolloi to examine the effects of inhibition of extracellular vs total (extracellular and intracellular) carbonic anhydrase (CA) activity on pulmonary and branchial/cutaneous gas transfer. In contrast to previous studies on Protopterus, which showed that the vast majority of CO(2) is excreted into the water through the gill and/or skin whereas O(2) uptake largely occurs via the lung, P. dolloi appeared to use the lung for the bulk of both O(2) uptake (91.0+/-2.9%) and CO(2) excretion (76.0+/-6.6%). In support of the lung as the more important site of CO(2) transfer, aerial hypercapnia (P(CO(2))=40 mmHg) caused a significant rise in partial pressure of arterial blood CO(2) (Pa(CO(2))) whereas a similar degree of aquatic hypercapnia was without effect on Pa(CO(2)). Intravascular injection of low levels (1.2 mg kg(-1)) of the slowly permanent CA inhibitor, benzolamide, was without effect on red blood cell CA activity after 30 min, thus confirming its suitability as a short-term selective inhibitor of extracellular CA. Benzolamide treatment did not affect CO(2) excretion, blood acid-base status or any other measured variable within the 30 min measurement period. Injection of the permeant CA inhibitor acetazolamide (30 mg kg(-1)) resulted in the complete inhibition of red cell CA activity within 10 min. However, CO(2) excretion (measured for 2 h after injection) and arterial blood acid-base status (assessed for 24 h after injection) were unaffected by acetazolamide treatment. Intra-arterial injection of bovine CA (2 mg kg(-1)) caused a significant increase in overall CO(2) excretion (from 0.41+/-0.03 to 0.58+/-0.03 mmol kg(-1) h(-1)) and an increase in air breathing frequency (from 19.0+/-1.3 to 24.7+/-1.8 breaths min(-1)) that was accompanied by a slight, but significant, reduction in Pa(CO(2)) (from 21.6+/-1.6 to 19.6+/-1.8 mmHg). The findings of this study are significant because they (i) demonstrate that, unlike in other species of African lungfish that have been examined, the gill/skin is not the major route of CO(2) excretion in P. dolloi, and (ii) suggest that CO(2) excretion in Protopterus may be less reliant on carbonic anhydrase than in most other fish species.

NO modulation of myocardial performance in fish hearts.

In the mammalian heart, intracardiac nitric oxide (NO) regulates in an autocrine-paracrine manner cardiac function in the beat-to-beat response (Starling's law of the heart), short-term response (phasic control, e.g. excitation-contraction coupling, responses to neurotransmitters and endocrines) and long-term response (tonic control by altering gene expression). This trio of NO temporal-dependent actions has a long evolutionary history, as we have documented in the prototypic vertebrate heart, the teleost heart. This heart shares a common structural and functional scenario with higher vertebrate hearts exhibiting, at the same time, differences in myoarchitecture (trabecular vs. compact type), blood supply (lacunary vs. vascular) and pumping performance (sensitivity to filling pressure), thus providing challenging opportunities for revealing aspects of unity and diversity of cardiac NO in vertebrates. Using in vitro working teleost heart preparations we have shown that, under basal conditions, NO through a cGMP-mediated mechanism modulates ventricular performance (negative inotropism) and remarkably increases the sensitivity to filling pressure (i.e. the Frank-Starling response). NO-cGMP mechanism also influences the short-term response elicited by inotropic agents such as acetylcholine and angiotensin II. A role of NO in long-term cardiac adaptation is illustrated by morphologic evidence (e.g. NOS immuno-localization in phylogenetically distant species) which emphasizes the importance of NO in reshaping the angio-myoarchitecture of the fish heart ventricle (i.e. compensation for regional heterogeneity). Finally, by studying the avascular hearts of teleosts and amphibians that lack vascular endothelium, a relevant role of endocardial endothelium-NO signalling in intracavitary regulation of myocardial performance has been firmly established, thus revealing its early evolutionary role in non-mammalian vertebrates.

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.