Nitrogen metabolism and branchial osmoregulatory acclimation in the juvenile marble goby, Oxyeleotris marmorata, exposed to seawater.

The marble goby, Oxyeleotris marmorata, considered a freshwater fish, was able to hypoosmoregulate successfully during 14 days of acclimation to seawater (30 per thousand) following 6 days of progressive increase in salinity. In seawater, there were slight perturbations in plasma osmolality and ionic concentrations, and significant increases in contents of some free amino acids, which presumably acted as osmolytes, in tissues. The muscle glutamine content increased significantly during seawater acclimation, and the activity and the protein abundance of glutamine synthetase increased significantly in the liver of fish exposed to seawater for 14 days. Exposure to seawater for 14 days also resulted in branchial osmoregulatory acclimation. There were significant increases in the activity and the protein abundance of gill Na(+)/K(+)-ATPase, and protein abundance of gill Na(+):K(+):2Cl(-) cotransporter (NKCC). Immunofluorescence microscopy of branchial Na(+)/K(+)-ATPase-immunoreactive cells revealed that exposure to seawater led to increases in protein expression of apical cystic fibrosis transmembrane receptor-like chloride channel and basolateral NKCC. Overall, our results indicate that juvenile marble goby can acclimate to brackish water and subsequently to seawater, and prompt future studies on the effects of salinity on its growth and development which may have important application to the Asian marble goby aquaculture industry.

Ammonia toxicity and tolerance in the brain of the African sharptooth catfish, Clarias gariepinus.

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air-breather, and can survive on land during drought. The objective of this study was to elucidate the mechanism of acute ammonia toxicity in C. gariepinus, and to examine whether methionine sulfoximine [MSO; an inhibitor of glutamine synthetase (GS)] or MK801 [an antagonist of N-methyl d-aspartate type glutamate (NMDA) receptors] had protective effects against acute ammonia toxicity in this fish. After 48 h of exposure to a sublethal concentration (75 mmoll(-1)) of environmental ammonia, the brain glutamine and ammonia contents in C. gariepinus increased to 15 micromol g(-1) and 4 micromol g(-1), respectively. Thus, C. gariepinus detoxified ammonia to glutamine and could tolerate high levels of glutamine in its brain. After C. gariepinus was injected intraperitoneally with a sublethal dose of ammonium acetate (CH(3)COONH(4); 8 micromol g(-1) fish) followed with emersion, brain ammonia and glutamine contents increased continuously during the subsequent 24-h period, reaching 7 and 18 micromol g(-1), respectively, at hour 24. These results suggest that when confronted with acute ammonia toxicity, the survival of C. gariepinus was crucially determined by its high tolerance of ammonia and high capacity to detoxify ammonia to glutamine in the brain. For fish injected with a sublethal dose of CH(3)COONH(4) (10 micromol g(-1) fish) followed with immersion, there were transient but significant increases in brain ammonia and glutamine contents, which peaked at hour 2 (4 micromol g(-1)) and hour 6 (6 micromol g(-1)), respectively. From these results, it can be deduced that C. gariepinus accumulated glutamine in preference to ammonia in its brain. By contrast, for fish injected with a lethal dose (20 micromol g(-1) fish) of CH(3)COONH(4) followed with immersion, the brain ammonia content increased drastically to 10 micromol g(-1) after 30 min, while the brain glutamine content remained relatively low at 5 micromol g(-1). Therefore, it can be concluded that increased synthesis and accumulation of glutamine in the brain was not the major cause of death in C. gariepinus confronted with acute ammonia toxicity. The determining factor of acute ammonia toxicity appeared to be the rate of ammonia build-up in the brain. MK801 (2 microg g(-1) fish) had no protective effect on C. gariepinus injected with a lethal dose of CH(3)COONH(4) (20 micromol g(-1) fish) indicating that activation of NMDA receptors might not be involved. By contrast, the prior administration of MSO (100 microg g(-1) fish) reduced the mortality rate from 100% to 80% and at the same time prolonged the time of death significantly from 27 min to 48 min. However, the protective effect of MSO was apparently unrelated to the inhibition of glutamine synthetase and prevention of glutamine accumulation in the brain. Instead, MSO affected activities of glutamate dehydrogenase and alanine aminotransferase and suppressed the rate of ammonia build up in the brain of fish injected with a lethal dose of CH(3)COONH(4).

Properties and expression of Na+/K+-ATPase α-subunit isoforms in the brain of the swamp eel, Monopterus albus, which has unusually high brain ammonia tolerance.

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l(-1)) and accumulate ammonia to high concentrations in its brain (4.5 µmol g(-1)). Na(+)/K(+)-ATPase (Nka) is an essential transporter in brain cells, and since NH4(+) can substitute for K(+) to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K(+) specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH4(+) to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na(+)/K(+)-ATPase and Na(+)/NH4(+)-ATPase activities over a range of K(+)/NH4(+) concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l(-1) NH4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na(+)/NH4(+)-ATPase activities were significantly lower than the Na(+)/K(+)-ATPase activities assayed at various NH4(+)/K(+) concentrations. Furthermore, the effectiveness of NH4(+) to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K(+) specificity of K(+) binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.

Intestinal osmoregulatory acclimation and nitrogen metabolism in juveniles of the freshwater marble goby exposed to seawater.

The objective of this study was to elucidate the role of the intestine from juveniles of the marble goby, Oxyeleotris marmorata, during seawater (SW) exposure. It has been reported elsewhere that SW-exposed juvenile O. marmorata exhibits hypoosmotic and hypoionic regulation, with the induction of branchial Na(+)/K(+)-ATPase (NKA), Na(+):K(+):2Cl(-) cotransporter (NKCC), and cystic fibrosis transmembrane receptor-like chloride channels. Here, we report that SW exposure also led to significant increases in the activity and protein abundance of NKA in, and probably an increase in Na(+) uptake through, its intestine. Additionally, there was an increase in apical NKCC immunoreactivity in the intestinal epithelium, indicating that there could be increased Cl(-) uptake through the intestine. These results suggest that absorption of ions, and hence water, from the intestinal lumen could be an essential part of the osmoregulatory process in juvenile O. marmorata during exposure to SW. Furthermore, there were significant increases in the glutamate content, and the aminating activity and protein abundance of glutamate dehydrogenase (GDH) in the intestine of fish exposed to SW. Since the intestinal glutamine synthetase activity and protein abundance decreased significantly, and the intestinal glutamine content remained unchanged, in the SW-exposed fish, excess glutamate formed via increased GDH activity in the intestine could be channeled to other organs to facilitate the increased synthesis of amino acids. Taken together, our results indicate for the first time that, besides absorbing ions and water during SW exposure, the intestine of juvenile O. marmorata also participated in altered nitrogen metabolism in response to salinity changes.

Acute ammonia toxicity and the protective effects of methionine sulfoximine on the swamp eel, Monopterus albus.

The objectives of this study were to examine how the swamp eel, Monopterus albus, defended against acute ammonia toxicity derived from the intraperitoneal injection with a sublethal dose (10 micromol g(-1) fish) of ammonium acetate (CH(3)COONH(4)) followed by 24 hr of emersion, and to elucidate the mechanisms of acute ammonia toxicity with respect to glutamine accumulation in the brain using L-methionine S-sulfoximine [MSO; a glutamine synthetase inhibitor]. When confronted with a sublethal dose of CH(3)COONH(4) followed by emersion, only a small fraction of the exogenous ammonia was excreted, and ammonia contents in various organs, especially the brain, increased transiently to high levels. Increased glutamine synthesis and decreased amino acid catabolism in and outside the brain were involved in the defence against acute ammonia toxicity. When injected with a lethal dose (16 micromol g(-1) fish) of CH(3)COONH(4) followed by emersion, ammonia (approximately 30 micromol g(-1) tissue), but not glutamine ( approximately 5 micromol g(-1) tissue), accumulated to extraordinarily high levels in the brain of succumbed fish. Hence, glutamine accumulation in the brain might not be the major mechanism of acute ammonia toxicity in M. albus. MSO (100 microg g(-1) fish) had a partial protective effect in fish injected with a lethal dose of CH(3)COONH(4). However, this effect was unrelated to the suppression of glutamine synthesis and accumulation in the brain. Instead, MSO suppressed the rate of ammonia buildup in the brain, possibly through its effects on glutamate dehydrogenase therein.

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.