Biomedical subjects

Shit F Chew

Publications and source records attributed to Shit F Chew.

At least 19 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

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

Greatly elevated urea excretion after air exposure appears to be carrier mediated in the slender lungfish (Protopterus dolloi).

Under aquatic conditions, Protopterus dolloi is ammoniotelic, excreting only small amounts of urea-N. However, upon return to water after 30 d estivation in air, the lungfish excretes only small amounts of ammonia-N but massive amounts of urea-N. A similar pattern is seen after 21-30 d of terrestrialization, a treatment in which the lungfish is air exposed but kept moist throughout. After both treatments, the time course of urea-N excretion is biphasic with an immediate increase, then a fall, and finally a second larger increase that peaks at about 12 h and may be prolonged for several days thereafter. Urea-N excretion rates during the second peak reach 2,000-6,000 micromol N kg(-1) h(-1), two to three orders of magnitude greater than rates in most fish and comparable only to rates in species known to employ UT-A type facilitated diffusion urea transporters. Divided chamber studies and measurements of the clearance rates of [3H]-PEG-4000 (a glomerular filtration and paracellular diffusion marker) and two structural analogs of urea ([14C]-acetamide and [14C]-thiourea) were performed to characterize the two peaks of urea-N excretion. The smaller first peak was almost equally partitioned between the head (including internal and external gills) and the body compartment (including urinary opening), was accompanied by only a modest increase in [14C]-acetamide clearance equal to that in [14C]-thiourea clearance, and could be accounted for by a large but short-lasting increase in [3H]-PEG-4000 clearance (to about fivefold the terrestrial rate). The delayed, much larger second peak in urea-N excretion represented an elevated efflux into both compartments but occurred mainly (72%) via the body rather than the head region. This second peak was accompanied by a substantial increase in [14C]-acetamide clearance but only a modest further rise in [14C]-thiourea clearance. The acetamide to thiourea permeability ratio was typical of UT-A type transporters in other fish. [3H]-PEG-4000 clearance was stable at this time at about double the terrestrial rate, and excretion rates of urea and its analogs were many fold greater than could be accounted for by [3H]-PEG-4000 clearance. We conclude that the first peak may be explained by elevated urinary excretion and paracellular diffusion across the gills upon resubmergence, while the second peak is attributable to a delayed and prolonged activation of a UT-A type facilitated diffusion mechanism, primarily in the skin and perhaps also in branchial epithelia.

Ammonia↗

PubMed2005

Changes in salinity and ionic compositions can act as environmental signals to induce a reduction in ammonia production in the African lungfish Protopterus dolloi.

The slender African lungfish, Protopterus dolloi, does not aestivate in a subterranean mud cocoon, but is capable of aestivating inside a layer of dried mucus on land during drought. In this study, we aimed to elucidate if a slight increase in salinity in association with changes in the ionic composition could act as signals for P. dolloi to decrease endogenous ammonia production, in preparation for aestivation when the external medium dries up. Specimens of P. dolloi exposed to 3 per thousand water for 6 days exhibited consistently lower daily urea excretion rate than the freshwater control. This led to significant decreases in the cumulative total nitrogenous wastes excreted on days 3, 5 and 6. On day 6, there were decreases in urea contents in various tissues and organs. Taken together, these results suggest that there was a decrease in the rate of urea synthesis, the magnitude of which was greater than the decrease in the rate of urea excretion, and therefore resulted in decreases in internal urea contents. A decrease in the rate of urea synthesis should result in a decrease in the rate of glutamine utilization, and subsequently led to the accumulations of glutamine and/or ammonia. However, there were no changes in contents of glutamine and ammonia in various tissues and organs in the experimental animals. A logical explanation for this is that there must be a simultaneous reduction in ammonia production; if not, ammonia would accumulate due to the decrease in rate of urea synthesis. Since fish were unfed during the experiment, endogenous ammonia must be derived mainly from amino acid catabolism. Therefore, these results suggest that a suppression of amino acid catabolism occurred in specimens exposed to 3 per thousand for 6 days. The differences in effects of freshwater and 3 per thousand water on endogenous ammonia production could not be due to food deprivation because both groups of fish were fasted for the same period. Because control and experimental fish were kept in water and because there were no changes in the wet mass of the fish and blood osmolality before and after the experiment, dehydration did not occur. Furthermore, both groups of fish have comparable blood pH, pO2 and pCO2 on day 6 as they had free access to air, and therefore CO2 retention could be eliminated as the initiating factor of suppressed endogenous ammonia production. In conclusion, our results suggest that P. dolloi could respond to increases in salinity and changes in ionic composition in the external medium by suppressing ammonia production in preparation for aestivation when the water dries up.

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

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

The giant mudskipper Periophthalmodon schlosseri facilitates active NH(4)(+) excretion by increasing acid excretion and decreasing NH(3) permeability in the skin.

Periophthalmodon schlosseri is an amphibious and obligatory air-breathing teleost, which is extremely tolerant to environmental ammonia. It actively excretes NH(4)(+) in ammonia loading conditions. For such a mechanism to operate efficaciously the fish must be able to prevent back flux of NH(3). P. schlosseri could lower the pH of 50 volumes (w/v) of 50% seawater in an artificial burrow from pH 8.2 to pH 7.4 in 1 day, and established an ambient ammonia concentration of 10 mmol l(-1) in 8 days. It could alter the rate of titratable acid efflux in response to ambient pH. The rate of net acid efflux (H(+) excretion) in P. schlosseri was pH-dependent, increasing in the order pH 6.0<7.0<8.0<8.5. Net acid flux in neutral or alkaline pH conditions was partially inhibited by bafilomycin, indicating the possible involvement of a V-type H(+)-ATPase. P. schlosseri could also increase the rate of H(+) excretion in response to the presence of ammonia in a neutral (pH 7.0) external medium. Increased H(+) excretion in P. schlosseri occurred in the head region where active excretion of NH(4)(+) took place. This would result in high concentrations of H(+) in the boundary water layer and prevent the dissociation of NH(4)(+), thus preventing a back flux of NH(3) through the branchial epithelia. P. schlosseri probably developed such an 'environmental ammonia detoxification' capability because of its unique behavior of burrow building in the mudflats and living therein in a limited volume of water. In addition, the skin of P. schlosseri had low permeability to NH(3). Using an Ussing-type apparatus with 10 mmol l(-1) NH(4)Cl and a 1 unit pH gradient (pH 8.0 to 7.0), the skin supported only a very small flux of NH(3) (0.0095 micromol cm(-2) min(-1)). Cholesterol content (4.5 micromol g(-1)) in the skin was high, which suggests low membrane fluidity. Phosphatidylcholine, which has a stabilizing effect on membranes, constituted almost 50% of the skin phospholipids, with phosphatidyleserine and phsophatidylethanolamine contributing only 13% and 15%, respectively. More importantly, P. schlosseri increased the cholesterol level (to 5.5 micromol g(-1)) and altered the fatty acid composition (increased total saturated fatty acid content) in its skin lipid after exposure to ammonia (30 mmol l(-1) at pH 7.0) for 6 days. These changes might lead to an even lower permeability to NH(3) in the skin, and reduced back diffusion of the actively excreted NH(4)(+) as NH(3) or the net influx of exogenous NH(3), under such conditions.

Acids↗

PubMed2004

Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land.

This study aimed to elucidate the strategies adopted by the African slender lungfish, Protopterus dolloi, to ameliorate the toxicity of ammonia during short (6 days) or long (40 days) periods of aestivation in a layer of dried mucus in open air in the laboratory. Despite decreases in rates of ammonia and urea excretion, the ammonia content in the muscle, liver, brain and gut of P. dolloi remained unchanged after 6 days of aestivation compared with the control fasted for 6 days. For specimens aestivated for 40 days, the ammonia contents in the muscle, liver and gut were significantly lower than those of the control fasted for 40 days, which suggests a decrease in the rate of ammonia production. In addition, there were significant increases in contents of alanine, aspartate and glutamate in the muscle, which suggests decreases in their catabolism. During the first 6 days and the last 34 days of aestivation, the rate of ammonia production was reduced to 26% and 28%, respectively, of the control rate (6.83 micromol day(-1) g(-1) on day 0). During the first 6 days and the next 34 days of aestivation, the averaged urea synthesis rate was 2.39-fold and 3.8-fold, respectively, greater than the value of 0.25 micromol day(-1) g(-1) for the day 0 control kept in water. No induction of activities of the ornithine-urea cycle (OUC) enzymes was observed in specimens aestivated for 6 days, because the suppression of ammonia production led to a light demand on the OUC capacity. For specimens aestivated for 40 days, the activities of carbamoyl phosphate synthetase, ornithine transcarbamylase and argininosuccinate synthetase + lyase were significantly greater than those of the control fasted for 40 days. This is in agreement with the observation that the rate of urea synthesis in the last 34 days was greater than that in the first 6 days of aestivation. P. dolloi aestivated in a thin layer of dried mucus in open air with high O(2) tension throughout the 40 days of aestivation, which could be the reason why it was able to sustain a high rate of urea synthesis despite this being an energy-intensive process. Our results indicate that a reduction in ammonia production and decreases in hepatic arginine and cranial tryptophan contents are important facets of aestivation in P. dolloi.

Africa↗

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 snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism.

The freshwater snakehead Channa asiatica is an obligatory air-breather that resides in slow-flowing streams and in crevices near riverbanks in Southern China. In its natural habitat, it may encounter bouts of aerial exposure during the dry seasons. In the laboratory, the ammonia excretion rate of C. asiatica exposed to terrestrial conditions in a 12 h:12 h dark:light regime was one quarter that of the submerged control. Consequently, the ammonia contents in the muscle, liver and plasma increased significantly, and C. asiatica was able to tolerate quite high levels of ammonia in its tissues. Urea was not the major product of ammonia detoxification in C. asiatica, which apparently did not possess a functioning ornithine urea cycle. Rather, alanine increased fourfold to 12.6 micromol g(-1) in the muscle after 48 h of aerial exposure. This is the highest level known in adult teleosts exposed to air or an ammonia-loading situation. The accumulated alanine could account for 70% of the deficit in ammonia excretion during this period, indicating that partial amino acid catabolism had occurred. This would allow the utilization of certain amino acids as energy sources and, at the same time, maintain the new steady state levels of ammonia in various tissues, preventing them from rising further. There was a reduction in the aminating activity of glutamate dehydrogenase from the muscle and liver of specimens exposed to terrestrial conditions. Such a phenomenon has not been reported before and could, presumably, facilitate the entry of alpha-ketoglutarate into the Krebs cycle instead of its amination to glutamate, as has been suggested elsewhere. However, in contrast to mudskippers, C. asiatica was apparently unable to reduce the rates of proteolysis and amino acid catabolism, because the reduction in nitrogenous excretion during 48 h of aerial exposure was completely balanced by nitrogenous accumulation in the body. Alanine accumulation also occurred in specimens exposed to terrestrial conditions in total darkness, with no change in the total free amino acid content in the muscle. Exercise on land led to a decrease in glycogen content, and an increase in lactate levels, with no significant effect on ammonia and alanine contents in the muscle of C. asiatica. Hence, unlike the mudskipper Periophthalmodon schlosseri, C. asiatica was incapable of increasing the rate of partial amino acid catabolism to sustain locomotory activities on land. Alanine formation therefore appears to be a common strategy adopted by obligatory air-breathing fishes to avoid ammonia toxicity (not a strategy to detoxify ammonia) on land, but not all of them can utilize it to fuel muscular activities.

Aerobiosis↗

PubMed2003

The swamp eel Monopterus albus reduces endogenous ammonia production and detoxifies ammonia to glutamine during 144 h of aerial exposure.

The swamp eel Monopterus albus inhabits muddy ponds, swamps, canals and rice fields, where it can burrow within the moist earth during the dry summer season, thus surviving for long periods without water. This study aimed to elucidate the strategies adopted by M. albus to defend against endogenous ammonia toxicity when kept out of water for 144 h (6 days). Like any other fish, M. albus has difficulties in excreting ammonia during aerial exposure. In fact, the rates of ammonia and urea excretions decreased significantly in specimens throughout the 144 h of aerial exposure. At 144 h, the ammonia and urea excretion rates decreased to 20% and 25%, respectively, of the corresponding control values. Consequently, ammonia accumulated to high levels in the tissues and plasma of the experimental specimens. Apparently, M. albus has developed relatively higher ammonia tolerance at the cellular and subcellular levels compared with many other teleost fish. Since the urea concentration in the tissues of specimens exposed to air remained low, urea synthesis was apparently not adopted as a strategy to detoxify endogenous ammonia during 144 h of aerial exposure. Instead, ammonia produced through amino acid catabolism was detoxified to glutamine, leading to the accumulation of glutamine in the body during the first 72 h of aerial exposure. Complementing the increased glutamine formation was a significant increase in glutamine synthetase activity in the liver of specimens exposed to air for 144 h. Formation of glutamine is energetically expensive. It is probably because M. albus remained relatively inactive on land that the reduction in energy demand for locomotory activity facilitated its exploitation of glutamine formation to detoxify endogenous ammonia. There was a slight decrease in the glutamine level in the body of the experimental animals between 72 h and 144 h of aerial exposure, which indicates that glutamine might not be the end product of nitrogen metabolism. In addition, these results suggest that suppression of endogenous ammonia production, possibly through reductions in proteolysis and amino acid catabolism, acts as the major strategy to avoid ammonia intoxication in specimens exposed to air for >/=72 h. It is concluded that glutamine formation and reduction in ammonia production together served as effective strategies to avoid the excessive accumulation of ammonia in the body of M. albus during 144 h of aerial exposure. However, these strategies might not be adequate to sustain the survival of M. albus in the mud for longer periods during drought because ammonia and glutamine concentrations had already built up to high levels in the body of specimens exposed to air for 144 h.

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↗