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

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

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

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

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

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

Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish Clarias batrachus.

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

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

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