Biomedical subjects

Wai P Wong

Publications and source records attributed to Wai P Wong.

17 recordsLinked to original sources

PubMed2006

Postprandial increases in nitrogenous excretion and urea synthesis in the Chinese soft-shelled turtle, Pelodiscus sinensis.

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

Ammonia↗

PubMed2006

Light induces an increase in the pH of and a decrease in the ammonia concentration in the extrapallial fluid of the giant clam Tridacna squamosa.

The objective of this study was to examine whether 12 h of light exposure would lead to an increase in the pH of and a decrease in the concentration of total ammonia in the extrapallial fluid of the giant clam Tridacna squamosa. We also aimed to elucidate indirectly whether movements of ammonia and/or protons (H(+)) occurred between the extrapallial fluid and the outer mantle epithelium. The pH of the extrapallial fluid of T. squamosa exposed to 12 h of light was significantly higher than that of clams exposed to 12 h of darkness. Conversely, the total ammonia concentration in the extrapallial fluid of the former was significantly lower than that of the latter. In addition, the glutamine content in the mantle adjacent to the extrapallial fluid of clams exposed to 12 h of light was significantly greater than that of clams exposed to 12 h of darkness. These results suggest that in the extrapallial fluid of T. squamosa exposed to light, NH(3) combined with H(+) as NH(+)(4) and that NH(+)(4) was transported into the mantle and used as a substrate for glutamine formation. Injection of NH(4)Cl into the extrapallial fluid led to an instantaneous increase in the total ammonia concentration therein, but the total ammonia concentration decreased subsequently and returned to the control value within 1 h. This is in support of the proposition that NH(+)(4) could be transported from the extrapallial fluid to the mantle. Injection of HCl into the extrapallial fluid led to an instantaneous decrease in the pH of the extrapallial fluid. However, there was a significant increase in pH within 1 h in light or darkness, achieving a partial recovery toward the control pH value. The increase in pH within this 1-h period in light or darkness was accompanied by a significant decrease in the total ammonia concentration in the extrapallial fluid, which supports the proposition that H(+) could be transported in combination with NH(3) as NH(+)(4). Therefore, our results prompt a reexamination of the previous proposition that the removal of H(+) by NH(3) can facilitate calcification in molluscs in general and an investigation of the relationship between H(+) removal through NH(+)(4) transport and light-enhanced calcification in T. squamosa.

Ammonia↗

PubMed2006

Microanatomy and ultrastructure of the kidney of the African lungfish Protopterus dolloi.

The Dipnoi (lungfishes) have developed true lungs, having the ability to take oxygen from both the gills and the lungs. During the tropical dry season, many lungfish estivate on land, breathing only air. The estivation period is accompanied by profound functional modifications, including the suppression of urine. Thus, the lungfish kidney must be designed to cope with these dramatic cyclic changes in renal function. We study here the microanatomy and the structure of the kidney of the African lungfish Protopterus dolloi, maintained under controlled freshwater conditions. Chemical microdissection, light microscopy, and scanning and transmission electron microscopy have been used. The nephrons of P. dolloi are composed of a renal corpuscle (RC) and of a renal tubule that appears divided into five morphologically distinct segments: neck segment (NS), proximal tubule (PT), intermediate segment (IS), distal tubule (DT), and collecting tubule (CT). Paired CTs abut into a collecting duct, the latter emptying into an archinephric duct. The RCs lie in the mid-zone of the kidney, between the PTs and the convoluted DTs. The spatial distribution of these elements allows recognition of a kidney zonation. The RCs group into clusters (3-4 RCs per cluster) that are supplied by a single arteriole surrounded by pericytes. Each cluster appears to represent a functional unit with a common hemodynamic regulatory mechanism. The major processes of the podocytes form flattened networks that appear to constitute an integrated system due to the presence of gap junctions. The existence of mesangial cells with large cell processes, and of mesangial cells with a dendritic appearance, suggests a complex functional role (contractile and phagocytic) for the mesangium. The NS and the IS are the narrowest nephron segments, formed only by multiciliated cells. The PT and the DT can be subdivided, based on the tubular morphology and on cell composition, into portions I and II: PTI is formed only by brush border (BB) cells, while PTII contains BB and multiciliated cells. The DTI is formed by segment-specific cells, while the DTII contains segment-specific and a small number of flask cells. The CT contains principal and flask cells in a 5:1 ratio. The flask cells adopt two different configurations (with a narrow canaliculus or with a large cavity). The main goal of this study was to disclose specific kidney features that could be related to function, phylogeny, and habitat. In addition, the present results constitute the basis for a study of the morphologic changes that should occur in the kidney of P. dolloi during estivation.

Animals↗

PubMed2006

Exposure to brackish water, upon feeding, leads to enhanced conservation of nitrogen and increased urea synthesis and retention in the Asian freshwater stingray Himantura signifer.

The white-edge freshwater whip ray Himantura signifer is ammonotelic in freshwater, but retains the capacities of urea synthesis and ureosmotic osmoregulation to survive in brackish water. The first objective of this study was to examine whether exposure to brackish water would lead to increases in food intake, and/or conservation of nitrogen in H. signifer upon daily feeding. Results obtained showed that a progressive increase in ambient salinity, from 1 per thousand to 15 per thousand over a 10-day period, did not lead to an increase in daily food intake. However, there were significant reductions in daily rates of ammonia and urea excretion in H. signifer during salinity changes, especially between day 5 (in 10 per thousand water) and day 10 (in 15 per thousand water) when compared to those of the control kept in 1 per thousand water. Consequently, there was a significant decrease in the percentage of nitrogen (N) from the food being excreted as nitrogenous waste (ammonia-N+urea-N) during this period. On day 10, the tissue urea contents in fish exposed to 15 per thousand water were significantly greater than those of fish kept in 1 per thousand water, and the excess urea-N accumulated in the former fish could totally account for the cumulative deficit in excretion of urea-N+ammonia-N during the 10-day period. Thus, it can be concluded that H. signifer is N-limited, and conserved more N from food when exposed to brackish water. The conserved N was converted to urea, which was retained in tissues for osmoregulation. The second objective of this study was to elucidate whether the retention of the capacity of N conservation in H. signifer would lead to an accumulation of urea in fish exposed to not only 15 per thousand water, but also 1 per thousand water, upon feeding. For fish pre-acclimated to 1 per thousand water or 15 per thousand water for 10 days and then fasted for 48 h, the rate of ammonia excretion in fish exposed to 15 per thousand water was consistently lower than that of fish exposed to 1 per thousand water, throughout the 36-h post-feeding period. In addition, the hourly rate of urea excretion in the former was significantly lower than that of the latter between hours 12 and 36. There were postprandial increases in ammonia contents in the muscle, liver, stomach, intestine, brain and plasma of fish kept in 1 per thousand water; but postprandial increases in ammonia occurred only in the liver and brain of fish exposed to 15 per thousand water, and the magnitudes of increases in the latter were smaller than those in the former. Indeed, postprandial increases in tissue urea contents occurred in both groups of fish, but the greatest increase in urea content was observed in the muscle of fish exposed to 15 per thousand water. Taken together, these results indicate that H. signifer in freshwater could be confronted with postprandial osmotic stress because of its capacity of conserving N and increasing urea synthesis upon feeding.

Adaptation, Physiological↗

PubMed2006

Active ammonia transport and excretory nitrogen metabolism in the climbing perch, Anabas testudineus, during 4 days of emersion or 10 minutes of forced exercise on land.

The climbing perch, Anabas testudineus, inhabits large rivers, canals, stagnant water bodies, swamps and estuaries, where it can be confronted with aerial exposure during the dry season. This study aimed to examine nitrogen excretion and metabolism in this fish during 4 days of emersion. Contrary to previous reports, A. testudineus does not possess a functional hepatic ornithineurea cycle because no carbamoyl phosphate synthetase I or III activity was detected in its liver. It was ammonotelic in water, and did not detoxify ammonia through increased urea synthesis during the 4 days of emersion. Unlike many air-breathing fishes reported elsewhere, A. testudineus could uniquely excrete ammonia during emersion at a rate similar to or higher than that of the immersed control. In spite of the fact that emersion had no significant effect on the daily ammonia excretion rate, tissue ammonia content increased significantly in the experimental fish. Thus, it can be concluded that 4 days of emersion caused an increase in ammonia production in A. testudineus, and probably because of this, a transient increase in the glutamine content in the brain occurred. Because there was a significant increase in the total essential free amino acid in the experimental fish after 2 days of emersion, it can be deduced that increased ammonia production during emersion was a result of increased amino acid catabolism and protein degradation. Our results provide evidence for the first time that A. testudineus was able to continually excrete ammonia in water containing 12 mmol l(-1) NH4Cl. During emersion, active ammonia excretion apparently occurred across the branchial and cutaneous surfaces, and ammonia concentrations in water samples collected from these surfaces increased to 20 mmol l(-1). It is probable that the capacities of air-breathing and active ammonia excretion facilitated the utilization of amino acids by A. testudineus as an energy source to support locomotor activity during emersion. As a result, it is capable of wandering long distance on land from one water body to another as reported in the literature.

Amino Acids↗

PubMed2005

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.

Acclimatization↗

PubMed2005

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.

Amino Acids↗

PubMed2005

Heart inflow tract of the African lungfish Protopterus dolloi.

We report a morphologic study of the heart inflow tract of the African lungfish Protopterus dolloi. Attention was paid to the atrium, the sinus venosus, the pulmonary vein, and the atrioventricular (AV) plug, and to the relationships between all these structures. The atrium is divided caudally into two lobes, has a common part above the sinus venosus, and appears attached to the dorsal wall of the ventricle and outflow tract through connective tissue covered by the visceral pericardium. The pulmonary vein enters the sinus venosus and runs longitudinally toward the AV plug. Then it fuses with the pulmonalis fold and disappears as an anatomic entity. However, the oxygenated blood is directly conveyed into the left atrium by the formation of a pulmonary channel. This channel is formed cranially by the pulmonalis fold, ventrally by the AV plug, and caudally and dorsally by the atrial wall. The pulmonalis fold appears as a wide membranous fold which arises from the left side of the AV plug and extends dorsally to form the roof of the pulmonary channel. The pulmonalis fold also forms the right side of the pulmonary channel and sequesters the upper left corner of the sinus venosus from the main circulatory return. The AV plug is a large structure, firmly attached to the ventricular septum, which contains a hyaline cartilaginous core surrounded by connective tissue. The atrium is partially divided into two chambers by the presence of numerous pectinate muscles extended between the dorsal wall of the atrium and the roof of the pulmonary channel. Thus, partial atrial division is both internal and external, precluding the more complete division seen in amphibians. The present report, our own unpublished observations on other Protopterus, and a survey of the literature indicate that not only the Protopterus, but also other lungfish share many morphologic traits.

Animals↗

PubMed2005

Ventricle and outflow tract of the African lungfish Protopterus dolloi.

We report a morphologic study of the heart ventricle and outflow tract of the African lungfish Protopterus dolloi. The ventricle is saccular and appears attached to the anterior pericardial wall by a thick tendon. An incomplete septum divides the ventricle into two chambers. Both the free ventricular wall and the incomplete ventricular septum are entirely trabeculated. Only a thin rim of myocardium separates the trabecular system from the subepicardial space. The outflow tract consists of proximal, middle, and distal portions, separated by two flexures, proximal and distal. The proximal outflow tract portion is endowed with a layer of compact, well-vascularized myocardium. This portion is homologous to the conus arteriosus observed in the heart of most vertebrates. The middle and distal outflow tract portions are arterial-like, thus being homologous to the bulbus arteriosus. However, the separation between the muscular and arterial portions of the outflow tract is not complete in the lungfish. A thin layer of myocardium covers the arterial tissue, and a thin layer of elastic tissue underlies the conus myocardium. Two unequal ridges composed of loose connective tissue, the spiral and bulbar folds, run the length of the outflow tract. They form an incomplete division of the outflow tract, but fuse at the distal end. The two folds are covered by endocardium and contain collagen, elastin, and fibroblast-like cells. They appear to be homologous to the dextro-dorsal and sinistro-ventral ridges observed during the development of the avian and mammalian heart. Two to three rows of vestigial arterial-like valves appear in the dorsal and ventral aspects of the conus. These valves are unlikely to have a functional role. The possible functional significance of the "gubernaculum cordis," the thick tendon extending between the anterior ventricular surface and the pericardium, is discussed.

Animals↗

PubMed2005

Chronic and acute ammonia toxicity in mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti: brain ammonia and glutamine contents, and effects of methionine sulfoximine and MK801.

The objective of this study was to elucidate if chronic and acute ammonia intoxication in mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti, were associated with high levels of ammonia and/or glutamine in their brains, and if acute ammonia intoxication could be prevented by the administration of methionine sulfoximine [MSO; an inhibitor of glutamine synthetase (GS)] or MK801 [an antagonist of N-methyl D-aspartate type glutamate (NMDA) receptors]. For P. schlosseri and B. boddaerti exposed to sublethal concentrations (100 and 8 mmol l(-1) NH4Cl, respectively, at pH 7.0) of environmental ammonia for 4 days, brain ammonia contents increased drastically during the first 24 h, and they reached 18 and 14.5 micromol g(-1), respectively, at hour 96. Simultaneously, there were increases in brain glutamine contents, but brain glutamate contents were unchanged. Because glutamine accumulated to exceptionally high levels in brains of P. schlosseri (29.8 micromol g(-1)) and B. boddaerti (12.1 micromol g(-1)) without causing death, it can be concluded that these two mudskippers could ameliorate those problems associated with glutamine synthesis and accumulation as observed in patients suffering from hyperammonemia. P. schlosseri and B. boddaerti could tolerate high doses of ammonium acetate (CH3COONH4) injected into their peritoneal cavities, with 24 h LC50 of 15.6 and 12.3 micromol g(-1) fish, respectively. After the injection with a sublethal dose of CH3COONH4 (8 micromol g(-1) fish), there were significant increases in ammonia (5.11 and 8.36 micromol g(-1), respectively) and glutamine (4.22 and 3.54 micromol g(-1), respectively) levels in their brains at hour 0.5, but these levels returned to normal at hour 24. By contrast, for P. schlosseri and B. boddaerti that succumbed within 15-50 min to a dose of CH3COONH4 (15 and 12 micromol g(-1) fish, respectively) close to the LC50 values, the ammonia contents in the brains reached much higher levels (12.8 and 14.9 micromol g(-1), respectively), while the glutamine level remained relatively low (3.93 and 2.67 micromol g(-1), respectively). Thus, glutamine synthesis and accumulation in the brain was not the major cause of death in these two mudskippers confronted with acute ammonia toxicity. Indeed, MSO, at a dosage (100 microg g(-1) fish) protective for rats, did not protect B. boddaerti against acute ammonia toxicity, although it was an inhibitor of GS activities from the brains of both mudskippers. In the case of P. schlosseri, MSO only prolonged the time to death but did not reduce the mortality rate (100%). In addition, MK801 (2 microg g(-1) fish) had no protective effect on P. schlosseri and B. boddaerti injected with a lethal dose of CH3COONH4, indicating that activation of NMDA receptors was not the major cause of death during acute ammonia intoxication. Thus, it can be concluded that there are major differences in mechanisms of chronic and acute ammonia toxicity between brains of these two mudskippers and mammalian brains.

Acetates↗

PubMed2004

Postprandial increases in nitrogenous excretion and urea synthesis in the giant mudskipper Periophthalmodon schlosseri.

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the giant mudskipper, Periophthalmodon schlosseri, with special emphasis on the role of urea synthesis in ammonia detoxification. The ammonia and urea excretion rates of P. schlosseri increased 1.70- and 1.92-fold, respectively, within the first 3 h after feeding on guppies. Simultaneously, there were significant decreases in ammonia levels in the plasma and the brain, and in urea contents in the muscle and liver, of P. schlosseri at 3 h post-feeding. Thus, it can be concluded that P. schlosseri was capable of unloading ammonia originally present in some of its tissues in anticipation of ammonia released from the catabolism of excess amino acids after feeding. Subsequently, there were significant increases in urea content in the muscle, liver and plasma (1.39-, 2.17- and 1.62-fold, respectively) at 6 h post-feeding, and the rate of urea synthesis apparently increased 5.8-fold between 3 h and 6 h. Increased urea synthesis might have occurred in the liver of P. schlosseri because the greatest increase in urea content was observed therein. The excess urea accumulated in the body at 6 h was completely excreted between 6 and 12 h, and the percentage of waste-N excreted as urea-N increased significantly to 26% during this period, but never exceeded 50%, the criterion for ureotely, meaning that P. schlosseri remained ammonotelic after feeding. By 24 h, 62.7% of the N ingested by P. schlosseri was excreted, out of which 22.6% was excreted as urea-N. This is the first report on the involvement of increased urea synthesis and excretion in defense against ammonia toxicity in the giant mudskipper, and our results suggest that an ample supply of energy resources, e.g. after feeding, is a prerequisite for the induction of urea synthesis. Together, increases in nitrogenous excretion and urea synthesis after feeding effectively prevented a postprandial surge of ammonia in the plasma of P. schlosseri as reported previously for other fish species. Consequently, contrary to previous reports, there were significant decreases in the ammonia content of the brain of P. schlosseri throughout the 24 h period post-feeding, accompanied by a significant decrease in brain glutamine content between 12 h and 24 h.

Ammonia↗

PubMed2004

Exposure to air, but not seawater, increases the glutamine content and the glutamine synthetase activity in the marsh clam Polymesoda expansa.

Polymesoda expansa spends a considerable portion of its life exposed to air in mangrove swamps where salinity fluctuates greatly. Thus, the aim of this study was to evaluate the effects of aerial exposure (transfer from 10 per thousand brackish water directly to air) or salinity changes (transfer from 10 per thousand brackish water directly to 30 per thousand seawater) on nitrogen metabolism in P. expansa. We concluded that P. expansa is non-ureogenic because carbamoyl phosphate (CPS) III activity was undetectable in the adductor muscle, foot muscle, hepatopancreas and mantle when exposed to brackish water (control), seawater or air for 17 days. It is ammonotelic as it excretes nitrogenous wastes mainly as ammonia in brackish water or seawater. After transfer to seawater for 17 days, the contents of total free amino acids (TFAA) in the adductor muscle, foot muscle, hepatopancreas and mantle increased significantly. This could be related to an increase in protein degradation because exposure to seawater led to a greater rate of ammonia excretion on days 15 and 17, despite unchanged tissue ammonia contents. Alanine was the major free amino acid (FAA) in P. expansa. The contribution of alanine to the TFAA pool in various tissues increased from 43-48% in brackish water to 62-73% in seawater. In contrast, in clams exposed to air for 17 days there were no changes in alanine content in any of the tissues studied. Thus, the functional role of alanine in P. expansa is mainly connected with intracellular osmoregulation. Although 8.5-16.1% of the TFAA pool of P. expansa was attributable to glutamine, the glutamine contents in the adductor muscle, foot muscle, hepatopancreas and mantle were unaffected by 17 days of exposure to seawater. However, after exposure to air for 17 days, there were significant increases in ammonia content in all these tissues in P. expansa, accompanied by significant increases in glutamine content (2.9-, 2.5-, 4.5- and 3.4-fold, respectively). Simultaneously, there were significant increases in glutamine synthetase activities in the adductor muscle (1.56-fold) and hepatopancreas (3.8-fold). This is the first report on the accumulation of glutamine associated with an upregulation of glutamine synthetase in a bivalve species in response to aerial exposure, and these results reveal that the evolution of glutamine synthesis as a means for detoxification of ammonia first occurred among invertebrates.

Air↗

PubMed2003

The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro) stingrays.

The white-edge freshwater whip ray Himantura signifer can survive in freshwater (0.7 per thousand ) indefinitely or in brackish water (20 per thousand ) for at least two weeks in the laboratory. In freshwater, the blood plasma was maintained hyperosmotic to that of the external medium. There was approximately 44 mmol l(-1) of urea in the plasma, with the rest of the osmolality made up mainly by Na(+) and Cl(-). In freshwater, it was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea. Unlike the South American freshwater stingray Potamotrygon motoro, H. signifer has a functional ornithine-urea cycle (OUC) in the liver, with hepatic carbamoylphosphate synthetase III (CPS III) and glutamine synthetase (GS) activities lower than those of the marine blue-spotted fan tail ray Taeniura lymma. More importantly, the stomach of H. signifer also possesses a functional OUC, the capacity (based on CPS III activity) of which was approximately 70% that in the liver. When H. signifer was exposed to a progressive increase in salinity through an 8-day period, there was a continuous decrease in the rate of ammonia excretion. In 20 per thousand water, urea levels in the muscle, brain and plasma increased significantly. In the plasma, osmolality increased to 571 mosmol kg(-1), in which urea contributed 83 mmol l(-1). Approximately 59% of the excess urea accumulated in the tissues of the specimens exposed to 20 per thousand water was equivalent to the deficit in ammonia excretion through the 8-day period, indicating that an increase in the rate of urea synthesis de novo at higher salinities would have occurred. Indeed, there was an induction in the activity of CPS III in both the liver and stomach, and activities of GS, ornithine transcarbamoylase and arginase in the liver. Furthermore, there was a significant decrease in the rate of urea excretion during passage through 5 per thousand, 10 per thousand and 15 per thousand water. Although the local T. lymma in full-strength sea water (30 per thousand ) had a much greater plasma urea concentration (380 mmol l(-1)), its urea excretion rate (4.7 micromol day(-1) g(-1)) was comparable with that of H. signifier in 20 per thousand water. Therefore, H. signifer appears to have reduced its capacity to retain urea in order to survive in the freshwater environment and, consequently, it could not survive well in full-strength seawater.

Acclimatization↗

PubMed2003

A comparison of the effects of environmental ammonia exposure on the Asian freshwater stingray Himantura signifer and the Amazonian freshwater stingray Potamotrygon motoro.

The white-edge whip tail ray Himantura signifer inhabits a freshwater environment but has retained the capability to synthesize urea de novo through the arginine-ornithine-urea cycle (OUC). The present study aimed to elucidate whether the capacity of urea synthesis in H. signifer could be upregulated in response to environmental ammonia exposure. When H. signifer was exposed to environmental ammonia, fairly high concentrations of ammonia were accumulated in the plasma and other tissues. This would subsequently reduce the net influx of exogenous ammonia by reducing the NH(3) partial pressure gradient across the branchial and body surfaces. There was also an increase in the OUC capacity in the liver. Since the ammonia produced endogenously could not be excreted effectively in the presence of environmental ammonia, it was detoxified into urea through the OUC. In comparison, the South American freshwater stingray Potamotrygon motoro, which has lost the capability to synthesize urea de novo, was unable to detoxify ammonia to urea during ammonia loading. No increase in glutamine was observed in the various tissues of H. signifer exposed to environmental ammonia despite a significant increase in the hepatic glutamine synthetase activity. These results indicate that the excess glutamine formed was channelled completely into urea formation through carbamoyl phosphate synthetase III. It has been reported elsewhere that both urea synthesis and urea retention were upregulated in H. signifer exposed to 20 per thousand water for osmoregulatory purposes. By contrast, for H. signifer exposed to environmental ammonia in freshwater, the excess urea formed was excreted to the external medium instead. This suggests that the effectiveness of urea synthesis de novo as a strategy to detoxify ammonia is determined not simply by an increase in the capacity of urea synthesis but, more importantly, by the ability of the animal to control the direction (i.e. absorption or excretion) and rate of urea transport. Our results suggest that such a strategy began to develop in those elasmobranchs, e.g. H. signifer, that migrate into a freshwater environment from the sea but not in those permanently adapted to a freshwater environment.

Amino Acids↗

PubMed2003

Urea synthesis in the African lungfish Protopterus dolloi--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure.

Like the marine ray Taeniura lymma, the African lungfish Protopterus dolloi possesses carbamoyl phosphate III (CPS III) in the liver and not carbamoyl phosphate I (CPS I), as in the mouse Mus musculus or as in other African lungfish reported elsewhere. However, similar to other African lungfish and tetrapods, hepatic arginase of P. dolloi is present mainly in the cytosol. Glutamine synthetase activity is present in both the mitochondrial and cytosolic fractions of the liver of P. dolloi. Therefore, we conclude that P. dolloi is a more primitive extant lungfish, which is intermediate between aquatic fish and terrestrial tetrapods, and represents a link in the fish-tetrapod continuum. During 6 days of aerial exposure, the ammonia excretion rate in P. dolloi decreased significantly to 8-16% of the submerged control. However, there were no significant increases in ammonia contents in the muscle, liver or plasma of specimens exposed to air for 6 days. These results suggest that (1). endogenous ammonia production was drastically reduced and (2). endogenous ammonia was detoxified effectively into urea. Indeed, there were significant decreases in glutamate, glutamine and lysine levels in the livers of fish exposed to air, which led to a decrease in the total free amino acid content. This indirectly confirms that the specimen had reduced its rates of proteolysis and/or amino acid catabolism to suppress endogenous ammonia production. Simultaneously, there were significant increases in urea levels in the muscle (8-fold), liver (10.5-fold) and plasma (12.6-fold) of specimens exposed to air for 6 days. Furthermore, there was an increase in the hepatic ornithine-urea cycle (OUC) capacity, with significant increases in the activities of CPS III (3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more importantly, glutamine synthetase (2.2-fold). This is the first report on the upregulation of OUC capacity and urea synthesis rate in an African lungfish exposed to air. Upon re-immersion, the urea excretion rate increased 22-fold compared with that of the control specimen, which is the greatest increase among fish during emersion-immersion transitions and suggests that P. dolloi possesses transporters that facilitate the excretion of urea in water.

Air↗

PubMedDate not supplied

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.

Alanine↗

PubMedDate not supplied

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.

Amino Acids↗