Changes in free amino acid synthesis in the perfused liver of an air-breathing walking catfish, Clarias batrachus infused with ammonium chloride: a strategy to adapt under hyperammonia stress

The changes in the free amino acid (FAA) levels, the rate of efflux of FAAs from the perfused liver, and the activity of some enzymes related to amino acid metabolism such as glutamate dehydrogenase (GDH, both reductive amination and oxidative deamination), glutamine synthetase (GS), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were studied in the liver of a freshwater air-breathing teleost, the walking catfish, Clarias batrachus, perfused with 5 and 10 mM NH(4)Cl. The level of the various non-essential FAAs increased significantly, with a total increase of about 150%, which was accompanied by a significant increase of both ammonia and urea-N in the perfused liver both with 5 and 10 mM NH(4)Cl. The rate of efflux of these non-essential FAAs from the perfused liver also increased significantly with a total increase of about 115% and 160% at 5 and 10 mM NH(4)Cl, respectively. The activity of the mentioned amino acid metabolism-related enzymes in the perfused liver also got stimulated, except for GDH in the ammonia forming direction and ALT, under a higher ammonia load. The activity (both tissue and specific) of GDH in the glutamate forming direction increased maximally, followed by AST and GS in a decreasing order. Owing to these physiological adaptive strategies related to amino acid metabolism along with the presence of a functional and regulatory urea cycle (reported earlier), it is believed that this catfish is able to survive in very high ambient ammonia or in the air or in the mud during habitat drying.     

Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish<Emphasis Type="Italic">clarias batrachus</Emphasis>

In the present study, the possible role of ureogenesis to avoid the accumulation of toxic ammonia to a lethal level under hyper-ammonia stress was tested in the air-breathing walking catfishClarias batrachus by exposing the fish at 25 mM NH₄Cl for 7 days. Excretion of ammonia by the NH₄Cl-exposed fish was totally suppressed, which was accompanied by significant accumulation of ammonia in different body tissues. The walking catfish, which is otherwise predominantly ammoniotelic, turned totally towards ureotelism from ammoniotelism with a 5-to 6-fold increase of urea-N excretion during exposure to higher ambient ammonia. Stimulation of ureogenesis was accompanied with significant increase of some of the key urea cycle enzymes such as carbamyl phosphate synthetase (urea cycle-related), argininosuccinate synthetase and argininosuccinate lyase both in hepatic and non-hepatic tissues. Due to this unique physiological strategy of turning towards ureotelism from ammoniotelism via the induced urea cycle, this air-breathing catfish is able to survive in very high ambient ammonia, which they face in certain seasons of the year in the natural habitat.     

Role of ureogenesis in the mud-dwelled Singhi catfish (Heteropneustes fossilis) under condition of water shortage

The air-breathing Singhi catfish Heteropneustes fossilis was kept inside moist peat for 1 month mimicking their normal habitat in summer and the role of ureogenesis for their survival in a water-restricted condition was studied. The ammonia excretion rate by the mud-dwelled fish increased transiently between 6 and 12 h of re-immersion in water to approximately between eight and 10-fold, followed by a sharp decrease almost to the normal level at the later part of re-immersion. The urea-N excretion by the mud-dwelled fish increased to approximately 11-fold within 0-3 h of re-immersion, followed by a gradual decrease from 9 h onwards. The rate of urea-N excretion by the mud-dwelled fish, however, remained significantly higher (approx. threefold more) than the control fish even after 36-48 h of re-immersion. Although there was a significant increase of both ammonia and urea levels in the plasma and other tissues (except ammonia in the brain), the level of accumulation of urea was higher than ammonia in the mud-dwelled fish as indicated by the decrease in the ratio of ammonia: urea level in different tissues including the plasma. The activities (units/g tissue and /mg protein) of glutamine synthetase and three enzymes of the urea cycle, carbamyl phosphate synthetase, argininosuccinate synthetase and argininosuccinate lyase increased significantly in most of the tissues (except the brain) of the mud-dwelled fish as compared to the control fish. Higher accumulation of ammonia in vivo in the mud-dwelled Singhi catfish is suggested to be one of the major factors contributing to stimulation of ureogenesis. Due to this physiological adaptive strategy of ureogenesis, possibly along with other physiological adaptation(s), this air-breathing amphibious Singhi catfish is able to survive inside the moist peat for months in a water-restricted condition.     

Effect of alkalinity (pH 10) on ureogenesis in the air-breathing walking catfish,Clarias batrachus

Exposure of fish to alkaline conditions inhibits the rate of ammonia excretion, leading to ammonia accumulation and toxicity. The purpose of this study was to determine the role of ureogenesis via the urea cycle, to avoid the accumulation of ammonia to a toxic level during chronic exposure to alkaline conditions, for the air-breathing walking catfish, Clarias batrachus, where a full complement of urea cycle enzyme activity has been documented. The walking catfish can survive in water with a pH up to 10. At a pH of 10 the ammonia excretion rate by the walking catfish decreased by approximately 75% within 6 h. Although there was a gradual improvement of ammonia excretion rate by the alkaline-exposed fish, the rate remained 50% lower, even after 7 days. This decrease of ammonia excretion was accompanied by a significant accumulation of ammonia in plasma and body tissues (except in the brain). Urea-N excretion for alkaline-exposed fish increased 2.5-fold within the first day, which was maintained until day 3 and was then followed by a slight decrease to maintain a 2-fold increase in the urea-N excretion rate, even after 7 days. There was also a higher accumulation of urea in plasma and other body tissues (liver, kidney, muscle and brain). The activity of glutamine synthetase and three enzymes operating in the urea cycle (carbamyl phosphate synthetase, argininosuccinate synthetase, argininosuccinate lyase) increased significantly in hepatic and extra-hepatic tissue, such as the kidney and muscle in C. batrachus, during exposure to alkaline water. A significant increase in plasma lactate concentration noticed during alkaline exposure possibly helped in the maintenance of the acid-base balance. It is apparent that the stimulation of ureogenesis via the induced urea cycle is one of the major physiological strategies adopted by the walking catfish (C. batrachus) during chronic exposure to alkaline water, to avoid the in vivo accumulation of ammonia to a toxic level in body tissues and for the maintenance of pH homeostasis.     

Effects of peritoneal injection of NH4HCO3 on nitrogen excretion and metabolism in the swamp eel Monopterus albus-- increased ammonia excretion with an induction of glutamine synthetase activity

Monopterus albus has to deal with high environmental ammonia concentrations during dry seasons and agricultural fertilization in rice fields. In this study, NH4HCO3 (10 micromol per g fish) was injected into the peritoneal cavity of M. albus, raising the level of ammonia in the body, in order to elucidate the strategies involved in defense against the toxicity of exogenous ammonia. During the subsequent 24 h after NH4HCO3 injection, there was a significant increase in the ammonia excretion rate, which indicates that the main strategy adopted by M. albus was to remove the majority of the exogenous ammonia through enhanced ammonia excretion. Exogenous ammonia was not detoxified into urea for excretion or accumulation. Six hours post-injection of NH4HCO3, ammonia content in the tissues built up significantly, especially in the brain, which suggests that M. albus had high tolerance of ammonia toxicity at the cellular and sub-cellular levels. By hour 12 post-injection, there were significant increases in the activities of glutamine synthetase in the muscle, liver, and gut, accompanied by significant increases in glutamine contents in the muscle and the liver. There was also a significant increase in the glutamine content in the brain at hour 6 post-injection of NH4HCO3. These results confirm the capability of M. albus to detoxify ammonia through glutamine synthesis. Overall, injection of NH4HCO3 had only minor effects on the contents of FAAs, other than glutamine, in tissues of M. albus because the majority (70%) of the injected ammonia was excreted within the 24-h period.     

Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia

We assessed the possible upregulation of glutamine synthetase (GS) and typical 'fish type' carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish (Clarias batrachus) during exposure to 25 mM NH(4)Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine-urea cycle possibly through the involvement of cortisol.     

Mitochondrial citrulline synthesis from ammonia and glutamine in the liver of ureogenic air-breathing catfish, Clarias batrachus (Linnaeus)

The possible synthesis of citrulline, a rate limiting step for urea synthesis via the ornithine-urea cycle (OUC) in teleosts was tested both in the presence of ammonia and glutamine as nitrogen-donating substrates by the isolated liver mitochondria of ureogenic air-breathing walking catfish, C. batrachus. Both ammonia and glutamine could be used as nitrogen-donating substrates for the synthesis of citrulline by the isolated liver mitochondria, since the rate of citrulline synthesis was almost equal in presence of both the substrates. The citrulline synthesis by the isolated liver mitochondria requires succinate at a concentration of 0.1 mM as an energy source, and also requires the involvement of intramitochondrial carbonic anhydrase activity for supplying HCO3 as another substrate for citrulline synthesis. The rate of citrulline synthesis was further stimulated significantly by the isolated liver mitochondria of the fish after pre-exposure to 25 mM NH4Cl for 7 days. Due to possessing this biochemical adaptational strategy leading to the amelioration of ammonia toxicity mainly by channeling ammonia directly and/or via the formation of glutamine to the OUC, this air-breathing catfish could succeed in surviving in high external ammonia, which it faces in its natural habitat in certain seasons of the year.     

African sharptooth catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids but actively excretes ammonia during exposure to environmental ammonia

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.     

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.     

Expression of ornithine-urea cycle enzymes in early life stages of air-breathing walking catfish Clarias batrachus and induction of ureogenesis under hyper-ammonia stress

The air-breathing walking catfish Clarias batrachus is a potential ureogenic teleost with having a full complement of ornithine-urea cycle (OUC) enzymes expressed in various tissues. The present study was aimed at determining the pattern of nitrogenous waste excretion in the form of ammonia-N and urea-N along with the changes of tissue ammonia and urea levels, and the expression of OUC enzymes and glutamine synthetase (GSase) in early life stages of this teleost, and further, to study the possible induction of ureogenesis in 15-day old fry under hyper-ammonia stress. The ammonia and urea excretion was visible within 12 h post-fertilization (hpf), which increased several-fold until the yolk was completely absorbed by the embryo. Although all the early developing stages were primarily ammoniotelic, they also excreted significant amount of nitrogen (N) in the form of urea-N (about 35-40% of total N). Tissue levels of ammonia and urea also increased along with subsequent developmental stages at least until the yolk absorption stage. All the OUC enzymes and GSase were expressed within 4-12 hpf showing an increasing trend of activity for all the enzymes until 350 hpf. There was a significant increase of activity of GSase, carbamyl phosphate synthetase III (CPSase III) and argininosuccinate lyase enzymes (ASL), accompanied with significant increase of enzyme protein concentration of at least two enzymes (GSase and CPSase III) in the 15-day old fry following exposure to 10 mM NH4Cl as compared to respective controls kept in water over a period of 72 h. Thus, it appears that the OUC enzymes are expressed in early life stages of walking catfish like other teleosts, but at relatively high levels and remain expressed all through the life stages with a potential of stimulation of ureogenesis throughout the life cycle as a sort of physiological adaptation to survive and breed successfully under hyper-ammonia and various other environmental-related stresses.     

Nitrogen metabolism and excretion in the aquatic chinese soft-shelled turtle, Pelodiscus sinensis, exposed to a progressive increase in ambient salinity

This study aimed to determine effects of 6-day progressive increase in salinity from 1 per thousand to 15 per thousand on nitrogen metabolism and excretion in the soft-shelled turtle, Pelodiscus sinensis. For turtles exposed to 15 per thousand water on day 6, the plasma osmolality and concentrations of Na+, Cl- and urea increased significantly, which presumably decreased the osmotic loss of water. Simultaneously, there were significant increases in contents of urea, certain free amino acids (FAAs) and water-soluble proteins that were involved in cell volume regulation in various tissues. There was an apparent increase in proteolysis, releasing FAAs as osmolytes. In addition, there might be an increase in catabolism of certain amino acids, producing more ammonia. The excess ammonia was retained as indicated by a significant decrease in the rate of ammonia excretion on day 4 in 15 per thousand water, and a major portion of it was converted to urea. The rate of urea synthesis increased 1.4-fold during the 6-day period, although the capacity of the hepatic ornithine urea cycle remained unchanged. Urea was retained for osmoregulation because there was a significant decrease in urea excretion on day 4. Increased protein degradation and urea synthesis implies greater metabolic demands, and indeed turtles exposed to 15 per thousand water had significantly higher O2 consumption rate than the freshwater (FW) control. When turtles were returned from 15 per thousand water to FW on day 7, there were significant increases in ammonia (probably released through increased amino acid catabolism) and urea excretion, confirming that FAAs and urea were retained for osmoregulatory purposes in brackish water.     

Glucocorticoid receptors are involved in the regulation of pulsatile urea excretion in toadfish

The objectives of this study were to characterize the pattern of pulsatile urea excretion in the gulf toadfish in the wake of exogenous cortisol loading and to determine the receptors involved in the regulation of this mechanism. Toadfish were fitted with indwelling arterial catheters and were infused with isosmotic NaCl for 48 h after which fish were treated with cortisol alone, cortisol+peanut oil, cortisol+RU486 (a glucocorticoid receptor antagonist) or cortisol+spironolactone (a mineralocorticoid receptor antagonist). Upon cortisol loading, fish treated with cortisol alone, cortisol+oil or cortisol+spironolactone experienced a two- to threefold reduction in pulsatile urea excretion. This reduction was due to a decrease in urea pulse size with no effect on pulse frequency compared to values measured during the control NaCl infusion period. In addition, these fish showed an increase in plasma urea concentrations upon treatment. These apparent effects of cortisol treatment were abolished in fish treated with cortisol+RU486. In contrast, these fish showed an increase in pulsatile urea excretion mediated by a twofold increase in pulse size with no change in frequency. Likewise, fish treated with cortisol+RU486 showed a significant decrease in plasma urea concentrations over the course of the experiment. The findings of this study indicate that high levels of cortisol reduce pulsatile urea excretion by decreasing pulse size. In addition, it appears that glucocorticoid receptors and not mineralocorticoid receptors are involved in the regulation of the toadfish pulsatile urea excretion mechanism.Communicated by G. Heldmaier     

The African sharptooth catfish Clarias gariepinus can tolerate high levels of ammonia in its tissues and organs during four days of aerial exposure

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and can survive on land during drought. The objective of this study was to elucidate how C. gariepinus defends against ammonia toxicity when exposed to terrestrial conditions. During 4 d of aerial exposure, there was no accumulation of urea in its tissues, and the rate of urea excretion remained low. Thus, exposure to terrestrial conditions for 4 d did not induce ureogenesis or ureotely in C. gariepinus. Volatilization of NH(3) was not involved in excreting ammonia during aerial exposure. In addition, there were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus; nor were there any changes in the glutamine levels in these tissues. However, there were extraordinarily high levels of ammonia in the muscle (14 micromol g(-1)), liver (18 micromol g(-1)), and brain (11 micromol g(-1)) of fish exposed to terrestrial conditions for 4 d. This is the first report on a fish adopting high tolerance of ammonia in cells and tissues as the single major strategy to defend against ammonia toxicity during aerial exposure. At present, it is uncertain how C. gariepinus tolerates such high levels of ammonia, especially in its brain, but it can be concluded that, contrary to previous reports on two air-breathing catfishes (Clarias batrachus and Heteropneustes fossilis) from India, C. gariepinus does not detoxify ammonia to urea or free amino acids on land.     

Changes in tissue free amino acid contents, branchial Na+/K+ -ATPase activity and bimodal breathing pattern in the freshwater climbing perch, Anabas testudineus (Bloch), during seawater acclimation

This study aimed to examine effects of short- or long-term acclimation to brackish water or seawater on the climbing perch, Anabas testudineus, which is an aquatic air-breathing teleost living typically in freshwater. A. testudineus exhibits hypoosmotic and hypoinoic osmoregulation; the plasma osmolality, [Na+] and [Cl-] of fish acclimated to seawater were consistently lower than those of the external medium. However, during short-term (1 day) exposure to brackish water (15 per thousand) or seawater (30 per thousand), these three parameters increased significantly. There were also significant increases in tissue ammonia and urea contents, contents of certain free amino acids (FAAs) in the muscle, and rates of ammonia and urea excretion in the experimental fish. The accumulated FAAs might have a transient role in cell volume regulation. In addition, these results indicate that increases in protein degradation and amino acid catabolism had occurred, possibly providing energy for the osmoregulatory acclimation of the gills in fish exposed to salinity stress. Indeed, there was a significant increase in the branchial Na+/K+ -ATPase activity in fish exposed to seawater for a prolonged period (7 days), and the plasma osmolality, [Na+] and [Cl-] and the tissue FAA contents of these fish returned to control levels. More importantly, there was a significant increase in the dependence on water-breathing in fish acclimated to seawater for 7 days. This suggests for the first time that A. testudineus could alter its bimodal breathing pattern to facilitate the functioning of branchial Na+/K+ -ATPase for osmoregulatory purposes.     

Marine (Taeniura lymma) and freshwater (Himantura signifer) elasmobranchs synthesize urea for osmotic water retention

The objective of this study was to elucidate whether the marine blue-spotted fantail ray, Taeniura lymma, and the freshwater white-edge whip ray, Himantura signifer, injected with NH(4)Cl intraperitoneally would excrete the majority of the excess ammonia as ammonia per se to ameliorate ammonia toxicity despite being ureogenic. To examine the roles of urea and the ornithine-urea cycle, experimental fishes were exposed to salinity changes after being injected with NH(4)Cl. The ammonia excretion rates of the marine ray, T. lymma, injected with NH(4)Cl followed by exposure to seawater (30 per thousand) or diluted seawater (25 per thousand) increased 13-fold and 10-fold, respectively, within the first 3 h. Consequently, the respective percentage of nitrogenous wastes excreted as ammonia were 55% and 65% compared with 21% of the saline-injected control, indicating that T. lymma became apparently ammonotelic after injection with NH(4)Cl. By hour 6, large portions (70%-85%) of the ammonia injected into T. lymma exposed to seawater or diluted seawater had been excreted, and T. lymma excreted much more nitrogenous wastes (135%-180%), in excess of the ammonia injected into the fish, during the 24-h period. For T. lymma exposed to seawater, a small portion (30%) of the ammonia injected into the fish was detoxified to urea during the first 6 h, but there was an apparent suppression of urea synthesis thereafter, contributing partially to the large decrease (19%) in urea contents in its muscle at hour 24. A major contributing factor to the decrease in urea content was a reduction in ammonia production, as indicated by a large deficit between urea loss in the muscle and excess ammonia accumulated plus excess nitrogen excreted in the experimental fish. The freshwater ray, H. signifer, injected with NH(4)Cl followed by exposure to freshwater (0.7 per thousand) or brackish water (10 per thousand) was capable of excreting all the ammonia injected into the body, mainly as ammonia, within 12 h. Like T. lymma, it also excreted the injected ammonia mainly as ammonia during the first 3 h postinjection. During this period, the percentage of the injected ammonia excreted in fish exposed to brackish water (28.4%+/-4.6%) was significantly lower than those exposed to freshwater (56.1%+/-8.26%). In contrast, the percentage of nitrogenous wastes being excreted as urea in the former (38.4%) was significantly greater than that in the latter (14.1%). These results suggest that a portion of the ammonia injected into the fish was turned into urea, and urea synthesis was increased transiently in fish exposed to brackish water during the initial postinjection period. However, urea was not retained effectively by H. signifer. Taken together, these results suggest that the primary function of the ornithine-urea cycle in ureogenic marine and freshwater elasmobranchs is to synthesize urea for osmotic water retention and not for ammonia detoxification.     

Synthesis of glutamate and aspartate in rat brain synaptosomes

ATP and glutamine are the sources of endogenous ammonia in rat brain synaptosomes. The amount of endogenous ammonia formed from exogenous ATP is not sufficient to assure the maximum rate of aspartate and glutamate accumulation in the synaptosomes utilizing pyruvate + malate. Addition of exogenous NH4+ or depolarization of synaptosome plasma membranes with high K+ concentration led to a twofold increase in the rate of accumulation of these amino acids. This indicates that both exogenous and endogenous NH4+ is involved in the synthesis of aspartate and glutamate in nerve terminals. Accumulation of glutamate was stimulated by aminooxyacetate and inhibited by haloperidol which indicates that NH4+ is bound in the reaction catalysed by glutamate dehydrogenase. Endogenous oxaloacetate derived from pyruvate metabolism was the substrate for synthesis of aspartate. Additive inhibition of aspartate accumulation by fluorocitrate and (-) hydroxyacetate shows that, in addition to the tricarboxylic acid cycle, the reaction catalysed by ATP-citrate lyase serves in the synaptosomes as another source of oxaloacetate.     

The African lungfish, Protopterus dolloi, detoxifies ammonia to urea during environmental ammonia exposure

The African lungfish, Protopterus dolloi, was able to maintain a low level of blood plasma ammonia during exposure to high concentrations of environmental ammonia. After 6 d of exposure to 30 or 100 mM NH(4)Cl, the total ammonia concentrations in the blood plasma were 0.288 and 0.289 mM, respectively, which were only 1.7-fold greater than the control value of 0.163 mM. In addition, accumulation of ammonia occurred only in the muscle, but not in the liver. This was achieved in part through urea synthesis, as reflected by significant increases in urea contents in the muscle, liver, and plasma of the experimental animals. In contrast with plasma ammonia, the plasma urea concentrations of specimens exposed to 30 or 100 mM NH(4)Cl for 6 d increased 15.4-fold and 18.8-fold, respectively. Taken together, these results suggest that P. dolloi upregulated the rate of urea synthesis to detoxify ammonia during environmental ammonia exposure and that the increased rate of urea synthesis was fast enough to compensate for the rate of endogenous ammonia production plus the net influx of exogenous ammonia in these experimental animals. Simultaneously, there were increases in the rates of urea excretion in the experimental animals between day 2 and day 6 of environmental ammonia exposure. Interestingly, the rates of urea excretion in specimens exposed to 100 mM NH(4)Cl were lower than those exposed to 30 mM NH(4)Cl, despite the presumably greater load of ammonia to be detoxified to urea in the former situation. It would appear that P. dolloi was regulating the rate of urea excretion during ammonia exposure to retain urea, which might have some physiological functions under environmental stresses yet to be determined. There were decreases in the contents of glutamate, glutamine, and total free amino acids in the liver of the experimental animals, which indirectly suggest that a reduction in the rate of proteolysis and/or amino acid catabolism would have occurred that might lead to a decrease in ammonia production. Our results suggest that, unlike marine elasmobranchs and coelacanths, which synthesize and retain urea for osmoregulatory purposes, the ureogenic P. dolloi was adapted to synthesizing and excreting urea for the purpose of ammonia detoxification.     

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.     

Responses to reversed NH3 and NH4+ gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus):evidence for NH4+/H+ exchange in the teleost and the elasmobranch

Ammonia excretion rates of channel catfish, Ictalurus punctatus, little skate (Raja erinacea), and blue crab (Callinectes sapidus) were measured in experimental regimes which permitted simultaneous assessment of the partial pressure gradients for nonionized NH3 and the chemical concentration gradients of NH4+. Under conditions of low external ammonia, the average ammonia excretion was +295 microM kg-1 h-1 for catfish, +149 microM kg-1 h-1 for blue crabs, and +59 microM kg-1 h1 for skates with partial pressure gradients of +72.5 mu Torr, +413 mu Torr, and +24.4 mu Torr, respectively; and [NH4+] gradients of +189 microM l-1, +643 microM l-1, and +107 microM l-1 (positive indicating greater from inside to medium). When the external ammonia was increased to 1.15 mM l-1, both gradients were reversed, and the net ammonia movement was initially from the external water into all three species. In the catfish the inward movement ceased, however, and ammonia excretion eventually resumed in the face of reversed gradients of both NH3 partial pressure and [NH4+]. Unidirectional Na+ influx, indicative of a Na+/NH4+ exchange, did not increase. The ammonia data, changes in titratable acidity, and net apparent H+ efflux were all consistent with a linked extrusion of internal NH4+ for external H+. Incorporation of such an exchange into a computer simulation model of the ammonia equilibrium and exchange system duplicated the experimental data. Other hypotheses failed to match experimental data, or failed to predict internal ammonia levels lower than outside. In the crab, internal ammonia levels rose rapidly to concentrations and partial pressures above the external medium until excretion was reestablished, with no evidence of maintenance of a reversed gradient. In the skate, internal concentrations rose appreciably in the first hour and continued to rise for 6-8 h, with no resumption of ammonia excretion. The interspecies differences appear to be due at least partly to differences in ammonia permeability of the gills.     

Utilization of nitrogen from 15NH4Cl and [15N]alanine for urea synthesis in hepatocytes from fed and starved rats

Utilization of N from 15NH4Cl and [15N]alanine for urea synthesis in hepatocytes isolated from fed and 24 hr starved rats was investigated. In hepatocytes isolated from fed rats, 54 and 65% of the added [15N]ammonia was utilized for urea synthesis in the presence of 0.5 and 2.0 mM NH4Cl, respectively. This utilization of [15N]ammonia in hepatocytes from starved rats was 2-fold lower. The amount of urea synthetized from endogenous sources was, in the presence of 0.5 and 2.0 mM NH4Cl, about 44 and 60% higher than in the control conditions (without NH4Cl). The considerable amount of added ammonia (30-44%) was utilized in processes other than urea synthesis. Alanine markedly diminished the utilization of 15N from NH4Cl in hepatocytes from both fed and starved rats. In these conditions (NH4Cl present), alanine significantly increased the urea formation in hepatocytes from starved rats and failed to affect the urea production in hepatocytes from fed rats. On the basis of 15N determination, it was concluded that both NH4Cl and alanine caused an increase in the utilization of nitrogen from endogenous sources in rat hepatocytes. This conclusion is in contrast with the results based only on the changes in ammonia and urea concentrations.