Effects of exercise on nitrogen excretion, carbamoyl phosphate synthetase III activity and related urea cycle enzymes in muscle and liver tissues of juvenile rainbow trout (Oncorhynchus mykiss).

The purpose of this study was to determine if carbamoyl phosphate synthetase III (CPSase III) and related urea cycle enzyme activities in skeletal muscle tissue of juvenile rainbow trout (Oncorhynchus mykiss) increase during short- or long-term exercise, in parallel with changes in whole-body urea excretion rates. Urea excretion was elevated by 65% in fish that swam at high-speed (50 cm/s) vs. low-speed (20 cm/s) over a 2-h period, with no significant changes in CPSase III, ornithine transcarbamoylase or glutamine synthetase activities in muscle tissue. Fish that swam for 4 days at high-speed had higher rates of ammonia excretion and GSase activity in muscle and liver tissue relative to low-speed swimmers. Calculations showed that 47-53% of excreted urea, theoretically could be accounted for by total muscle CPSase III activity in juvenile and adult trout. The data indicate that increases in the rate of urea excretion during short-term high intensity exercise are not linked to higher activities of urea cycle enzymes in muscle tissue, but this does not rule out the possibility of increased flux through muscle CPSase III and related enzymes. Furthermore, these results indicate that urea cycle enzyme activities in skeletal muscle tissue can account for a significant portion of total urea excretion in juvenile and adult trout.     

Detection and basic properties of carbamoyl phosphate synthetase III during teleost ontogeny: a case study in the Atlantic halibut (Hippoglossus hippoglossus L.)

The presence of carbamoyl phosphate synthetase III (CPSase III), catalyzing the first step of the urea cycle in fish, in Atlantic halibut (Hippoglossus hippoglossus L.) yolk-sac larvae and adult white muscle has been established using gel filtration chromatography to separate the CPSase III from the pyrimidine-pathway related CPSase II. The results are consistent with the hypothesis that teleostean fish express urea cycle enzymes during early development and with recent observations of low levels of CPSase III in muscle tissue. The presence of CPSase III in crude extracts could not be established using sensitive assay conditions to discriminate between CPSase III and CPSase II. However, kinetic characterization after chromatographic separation identified each as typical CPSase II and CPSase III activities, respectively. The CPSase III was less sensitive to activation by N-acetyl- -glutamate and had a higher K_m for ammonia than CPSase III found in other species. These results suggest that precise quantitation of low levels of CPSase III in the presence of CPSase II by assaying crude extracts may be difficult unless the enzymes are first separated and the kinetic properties of CPSase III are determined; the results indicate that assaying larval extracts of Atlantic halibut in the presence of uridine triphosphate results in CPSase activity that reflects mostly CPSase III and can, therefore, be used to measure changes in CPSase III activity.     

Lamprey parasitism of sharks and teleosts: high capacity urea excretion in an extant vertebrate relic

We observed 10 sea lampreys (Petromyzon marinus) parasitizing basking sharks (Cetorhinus maximus), the world's second largest fish, in the Bay of Fundy. Due to the high concentrations of urea in the blood and tissues of ureosmotic elasmobranchs, we hypothesized that sea lampreys would have mechanisms to eliminate co-ingested urea while feeding on basking sharks. Post-removal urea excretion rates (J(Urea)) in two lampreys, removed from separate sharks by divers, were initially 450 ( approximately 9000 micromol N kg-1 h-1) and 75 times ( approximately 1500 micromol N kg-1 h-1) greater than basal (non-feeding) rates ( approximately 20 micromol N kg-1 h-1). In contrast, J(Urea) increased by 15-fold after parasitic lampreys were removed from non-ureosmotic rainbow trout (Oncorhynchus mykiss). Since activities of the ornithine urea cycle (OUC) enzymes, carbamoyl phosphate synthetase III (CPSase III) and ornithine carbamoyl transferase (OCT) were relatively low in liver and below detection in intestine and muscle, it is unlikely that the excreted urea arose from de novo urea synthesis. Measurements of arginase activity suggested that hydrolysis of dietary arginine made a minor contribution to J(Urea.). Post-feeding ammonia excretion rates (J(Amm)) were 15- to 25-fold greater than basal rates in lampreys removed from both basking sharks and rainbow trout, suggesting that parasitic lampreys have a high capacity to deaminate amino acids. We conclude that the sea lamprey's ability to penetrate the dermal denticle armor of sharks, to rapidly excrete large volumes of urea and a high capacity to deaminate amino acids, represent adaptations that have contributed to the evolutionary success of these phylogenetically ancient vertebrates.     

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.     

The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge

Urea synthesis via the hepatic ornithine urea cycle (OUC) has been well described in elasmobranchs, but it is unknown whether OUC enzymes are also present in extrahepatic tissues. Muscle and liver urea, trimethylamine oxide (TMAO), and other organic osmolytes, as well as selected OUC enzymes (carbamoyl phosphate synthetase III, ornithine transcarbamoylase, arginase, and the accessory enzyme glutamine synthetase), were measured in adult little skates (Raja erinacea) exposed to 100% or 75% seawater for 5 d. Activities of all four OUC enzymes were detected in the muscle. There were no changes in muscle OUC activities in skates exposed to 75% seawater; however, arginase activity was significantly lower in the liver, compared to controls. Urea, TMAO, and several other osmolytes were significantly lower in the muscle of little skates exposed to 75% seawater, whereas only glycerophosphorylcholine was significantly lower in the liver. Urea excretion rates were twofold higher in skates exposed to 75% seawater. Taken together, these data suggest that a functional OUC may be present in the skeletal muscle tissues of R. erinacea. As well, enhanced urea excretion rates and the downregulation of the anchor OUC enzyme, arginase, in the liver may be critical in regulating tissue urea content under dilute-seawater stress.     

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.     

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.     

Sequence, expression and evolutionary relationships of carbamoyl phosphate synthetase I in the toad Xenopus laevis

The sequence of carbamoyl phosphate synthetase I (CPSase I) cDNA and expression of the enzyme in liver of the toad Xenopus laevis are reported. CPSase I mRNA increases 6-fold when toads are exposed to high salinity for extended periods of time. The deduced 1,494-amino acid sequence of the CPSase I is homologous to other CPSases and reveals a domain structure and conserved amino acids common to other CPSases. A serine residue (S287) is present where there is a cysteine residue required for glutamine-dependent activity in CPSase Types III and II (Type I CPSases utilize only ammonia as nitrogen-donating substrate). A sequence of DNA 964 bases upstream from the ATG start codon for the CPSase I gene is also reported. Phylogenetic analysis for 30 CPSase isoforms, including X. laevis CPSase I, across a wide spectrum of phyla is reported and discussed. The results are consistent with the views that eukaryotic CPSase II as a multifunctional complex evolved from prokaryotic CPSase II and that CPSase I in terrestrial vertebrates and CPSase III in fishes arose from eukaryotic CPSase II by independent events after the divergence of plants in eukaryotic evolution.     

Excretory nitrogen metabolism in the juvenile axolotl Ambystoma mexicanum: differences in aquatic and terrestrial environments

The fully grown but nonmetamorphosed (juvenile) axolotl Ambystoma mexicanum was ureogenic and primarily ureotelic in water. A complete ornithine-urea cycle (OUC) was present in the liver. Aerial exposure impeded urea (but not ammonia) excretion, leading to a decrease in the percentage of nitrogen excreted as urea in the first 24 h. However, urea and not ammonia accumulated in the muscle, liver, and plasma during aerial exposure. By 48 h, the rate of urea excretion recovered fully, probably due to the greater urea concentration gradient in the kidney. It is generally accepted that an increase in carbamoyl phosphate synthetase activity is especially critical in the developmental transition from ammonotelism to ureotelism in the amphibian. Results from this study indicate that such a transition in A. mexicanum would have occurred before migration to land. Aerial exposure for 72 h exhibited no significant effect on carbamoyl phosphate synthetase-I activity or that of other OUC enzymes (with the exception of ornithine transcarbamoylase) from the liver of the juvenile A. mexicanum. This supports our hypothesis that the capacities of OUC enzymes present in the liver of the aquatic juvenile axolotl were adequate to prepare it for its invasion of the terrestrial environment. The high OUC capacity was further supported by the capability of the juvenile A. mexicanum to survive in 10 mM NH(4)Cl without accumulating amino acids in its body. The majority of the accumulating endogenous and exogenous ammonia was detoxified to urea, which led to a greater than twofold increase in urea levels in the muscle, liver, and plasma and a significant increase in urea excretion by hour 96. Hence, it can be concluded that the juvenile axolotl acquired ureotelism while submerged in water, and its hepatic capacity of urea synthesis was more than adequate to handle the toxicity of endogenous ammonia during migration to land.     

The crab-eating frog, Rana cancrivora, up-regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity

The crab-eating frog Rana cancrivora is one of only a handful of amphibians worldwide that tolerate saline waters. They typically inhabit brackish water of mangrove forests of Southeast Asia, but live happily in freshwater and can be acclimated to 75% seawater (25 ppt) or higher. We report here that after transfer of juvenile R. cancrivora from freshwater (1 ppt) to brackish water (10 -->20 or 20 -->25 ppt; 4-8 d) there was a significant increase in the specific activity of the key hepatic ornithine urea cycle enzyme (OUC), carbamoyl phosphate synthetase I (CPSase I). At 20 ppt, plasma, liver and muscle urea levels increased by 22-, 21-, and 11-fold, respectively. As well, muscle total amino acid levels were significantly elevated by 6-fold, with the largest changes occurring in glycine and beta-alanine levels. In liver, taurine levels were 5-fold higher in frogs acclimated to 20 ppt. There were no significant changes in urea or ammonia excretion rates to the environment. As well, the rate of urea influx (J(in) (urea)) and efflux (J(out) (urea)) across the ventral pelvic skin did not differ between frogs acclimated to 1 versus 20 ppt. Taken together, these findings suggest that acclimation to saline water involves the up-regulation of hepatic urea synthesis, which in turn contributes to the dramatic rise in tissue urea levels. The lack of change in urea excretion rates, despite the large increase in tissue-to-water gradients further indicates that mechanisms must be in place to prevent excessive loss of urea in saline waters, but these mechanisms do not include cutaneous urea uptake. Also, amino acid accumulation may contribute to an overall rise in the osmolarity of the muscle tissue, but relative to urea, the contribution is small.     

Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.     

Shifting patterns of nitrogen excretion and amino acid catabolism capacity during the life cycle of the sea lamprey (Petromyzon marinus)

The jawless fish, the sea lamprey (Petromyzon marinus), spends part of its life as a burrow-dwelling, suspension-feeding larva (ammocoete) before undergoing a metamorphosis into a free swimming, parasitic juvenile that feeds on the blood of fishes. We predicted that animals in this juvenile, parasitic stage have a great capacity for catabolizing amino acids when large quantities of protein-rich blood are ingested. The sixfold to 20-fold greater ammonia excretion rates (J(Amm)) in postmetamorphic (nonfeeding) and parasitic lampreys compared with ammocoetes suggested that basal rates of amino acid catabolism increased following metamorphosis. This was likely due to a greater basal amino acid catabolizing capacity in which there was a sixfold higher hepatic glutamate dehydrogenase (GDH) activity in parasitic lampreys compared with ammocoetes. Immunoblotting also revealed that GDH quantity was 10-fold and threefold greater in parasitic lampreys than in ammocoetes and upstream migrant lampreys, respectively. Higher hepatic alanine and aspartate aminotransferase activities in the parasitic lampreys also suggested an enhanced amino acid catabolizing capacity in this life stage. In contrast to parasitic lampreys, the twofold larger free amino acid pool in the muscle of upstream migrant lampreys confirmed that this period of natural starvation is accompanied by a prominent proteolysis. Carbamoyl phosphate synthetase III was detected at low levels in the liver of parasitic and upstream migrant lampreys, but there was no evidence of extrahepatic (muscle, intestine) urea production via the ornithine urea cycle. However, detection of arginase activity and high concentrations of arginine in the liver at all life stages examined infers that arginine hydrolysis is an important source of urea. We conclude that metamorphosis is accompanied by a metabolic reorganization that increases the capacity of parasitic sea lampreys to catabolize intermittently large amino acid loads arising from the ingestion of protein rich blood from their prey/hosts. The subsequent generation of energy-rich carbon skeletons can then be oxidized or retained for glycogen and fatty acid synthesis, which are essential fuels for the upstream migratory and spawning phases of the sea lamprey's life cycle.     

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.     

Contribution of dietary arginine to nitrogen utilisation and excretion in juvenile sea bass (Dicentrarchus labrax) fed diets differing in protein source

The role of dietary arginine in affecting nitrogen utilisation and excretion was studied in juvenile European sea bass (Dicentrarchus labrax) fed for 72 days with diets differing in protein sources (plant protein-based (PM) and fish-meal-based (FM)). Fish growth performance and nitrogen utilisation revealed that dietary Arg surplus was beneficial only in PM diets. Dietary Arg level significantly affected postprandial plasma urea concentrations. Hepatic arginase activity increased (P<0.05) in response to dietary Arg surplus in fish fed plant protein diets; conversely ornithine transcarbamylase activity was very low and inversely related to arginine intake. No hepatic carbamoyl phosphate synthetase III activity was detected. Dietary arginine levels did not affect glutamate dehydrogenase activity. A strong linear relationship was found between liver arginase activity and daily urea-N excretion. Dietary Arg excess reduced the proportion of total ammonia nitrogen excreted and increased the contribution of urea-N over the total N excretion irrespective of dietary protein source. Plasma and excretion data combined with enzyme activities suggest that dietary Arg degradation via hepatic arginase is a major pathway for ureagenesis and that ornithine-urea cycle is not completely functional in juvenile sea bass liver.     

Phylogenetic aspects of carbamoyl phosphate synthetase in lungfish: a transitional enzyme in transitional fishes

Carbamoyl phosphate synthetase (CPS) catalyses the formation of carbamoyl phosphate from glutamine or ammonia, bicarbonate and ATP. There are three different isoforms of CPS that play vital roles in two metabolic pathways, pyrimidine biosynthesis (CPS II) and arginine/urea biosynthesis (CPS I and CPS III). Gene duplication has been proposed as the evolutionary mechanism creating this gene family with CPS II likely giving rise to the CPS I/III clade. In the evolutionary history of this gene family it is still undetermined when CPS I diverged from CPS III on the path to terrestriality in the vertebrates. Transitional organisms such as lungfishes are of particular interest because they are capable of respiring via gills and with lungs and therefore can be found in both aquatic and terrestrial environments. Notably, enzymatic characterization of the mitochondrial CPS isoforms in this transitional group has not led to clear conclusions. In order to determine which CPS isoform is present in transitional animals, we examined partial sequences for liver CPS amplified from five species of lungfish, and a larger fragment of CPS from one lungfish species (Protopterus annectens) and compared them to CPS isoforms from other fish and mammals. Enzyme activities for P. annectens liver were also examined. While enzyme activities did not yield a clear distinction between isoforms (virtually equal activities were obtained for either CPS I or III), CPS sequences from the lungfishes formed a monophyletic clade within the CPS I clade and separate from the CPS III clade of other vertebrates. This finding implies that the mitochondrial isoform of CPS in lungfish is derived from CPS I and is likely to have a physiological function similar to CPS I. This finding is important because it supports the hypothesis that lungfish employ a urea cycle similar to terrestrial air-breathing vertebrates.     

Role of amino acid metabolism in an air-breathing catfish,Clarias batrachus in response to exposure to a high concentration of exogenous ammonia

The air-breathing ureogenic walking catfish (Clarias batrachus) faces various environmental constraints throughout the year leading to the problem of accumulation of toxic ammonia. In the present study, the possible role of conversion of accumulated ammonia to various non-essential free amino acids (FAAs) was tested in this fish under hyper-ammonia stress caused by exposing the fish at 25 mM NH(4)Cl for 7 days. Significant accumulation of ammonia of approximately two- to threefold was observed in different tissues (except in the brain), which was accompanied with the significant accumulation of non-essential FAAs in the NH(4)Cl-exposed fish. There was approximately two- to threefold increase of non-essential FAAs in different tissues and in the plasma of the NH(4)Cl-exposed fish compared to the control fish after 7 days of exposure, which was mainly attributable to the increase of Asp, Ala, Gly, Glu, Gln and taurine (Tau) concentrations in general, with certain tissue-specific variations. This was also accompanied with significant increase of activity of certain amino acid metabolism-related enzymes such as the glutamine synthetase (approx. two- to threefold), glutamate dehydrogenase (ammonia utilizing direction) (approx. twofold), aspartate and alanine aminotransaminases (approx. twofold) mainly in the liver, kidney and muscle of the NH(4)Cl-exposed fish. Thus, it appears that the walking catfish has the capacity of active conversion of accumulated ammonia to non-essential FAAs under condition of high concentrations of external ammonia. However, the increase of urea excretion rate due to active conversion of ammonia to urea via the induced urea cycle appears to be quantitatively much more important pathway than the increase of tissue levels of FAAs in dealing with a severe ammonia load.     

Decreased urea synthesis in cafeteria-diet-induced obesity in the rat.

Feeding rats with a cafeteria diet resulted in increases in total body weight and in epididymal-adipose-tissue weight. Those rats excreted significantly less N than did controls. The amount of N ingested by cafeteria-diet-fed rats was kept equal to that of controls. This decrease in N excretion is explained by a decrease in urinary excretion of urea. This may be due to the following facts. The rate of synthesis of urea from precursors by isolated hepatocytes from cafeteria-diet-fed rats was lower than in controls. In cafeteria-diet-fed rats the activities of all the enzymes of the urea cycle are decreased. The major percentage decreases are those of carbamoylphosphate synthetase (EC 6.3.4.16) and of argininosuccinate synthetase (EC 6.3.4.5), the enzymes probably involved in the regulation of the overall rate of the cycle. When rats are switched to normal chow diet, the enzyme activities return to normal values. The uptake of amino acids by liver of cafeteria-diet-fed rats is lower than in controls. These results contrast with those obtained previously by using other models of obesity in rat (i.e. genetic or hypothalamic), in which N excretion was increased.     

Nitrogen metabolism and excretion in Allenbatrachus grunniens(L): effects of variable salinity, confinement, high pH and ammonia loading

The nitrogen metabolism and excretion patterns of the grunting toadfish Allenbatrachus grunniens and the effects of salinity on these processes were examined. Individuals of A. grunniens were subjected to several experimental treatments, including variable salinity (2 to 30), high pH (8·5 compared to 7·0 for controls), high environmental ammonia (10 mM) and confinement to small water volumes, and measurements were made of activities of selected enzymes of nitrogen metabolism, ammonia and urea excretion rates, and tissue and plasma contents of ammonia, urea and amino acids. Activities of key ornithine‐urea cycle enzymes were rather low ( e.g . liver carbamoyl phosphate synthetase III activity was 0·001 μmols min⁻¹ g⁻¹), and A. grunniens consistently demonstrated a low capacity for urea excretion despite significant elevations of plasma and tissue ammonia contents by the high pH and high ammonia treatments. This species could thus be categorized as ammoniotelic. Total free amino acid contents in plasma and tissues were increased by the high pH and high ammonia treatments, but no patterns were discerned in individual amino acids that would indicate any preferential accumulation ( e.g . alanine and glutamine) as has been noted previously in several semi‐terrestrial fish species. Thus, it appeared that A. grunniens was not unusual in its patterns of nitrogen metabolism and excretion in comparison to other 'typical' teleosts. Furthermore, manipulation of salinity had no major effects on nitrogen excretion in either this species or in comparative studies with the ureotelic gulf toadfish Opsanus beta . The results are discussed in the context of the broader pattern of nitrogen metabolism and excretion in the Batrachoididae.