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 catfishClarias 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.

Both hypotonic exposure (185 mOsmol/l) and infusion of glutamine plus glycine (2 mmol/l each) along with the isotonic medium caused a significant increase of¹⁴CO₂ production from [1-¹⁴C]glucose by 110 and 70%, respectively, from the basal level of 18.4 ± 1.2 nmol/g liver/min from the perfused liver ofClarias batrachus. Conversely, hypertonic exposure (345 mOsmol/l) caused significant decrease of¹⁴CO₂ production from [1-¹⁴C]glucose by 34%.¹⁴CO₂ production from [6-¹⁴C]glucose was largely unaffected by anisotonicity. The steady-state release of oxidized glutathione (GSSG) into bile was 1.18 ±0.09 nmol/g liver/min, which was reduced significantly by 36% and 34%, respectively, during hypotonic exposure and amino acid-induced cell swelling, and increased by 34% during hypertonic exposure. The effects of anisotonicity on¹⁴CO₂ production from [1-¹⁴C]glucose and biliary GSSG release were also observed in the presence of t-butylhydroperoxide (50 (Amol/1). The oxidative stress-induced cell injury, caused due to infusion of t-butylhydroperoxide, was measured as the amount of lactate dehydrogenase (LDH) leakage into the effluent from the perfused liver; this was found to be affected by anisotonicity. Hypotonic exposure caused significant decrease of LDH release and hypertonic exposure caused significant increase of LDH release from the perfused liver. The data suggest that hypotonically-induced as well as amino acid-induced cell swelling stimulates flux through the pentose-phosphate pathway and decreases loss of GSSG under condition of mild oxidative stress; hypotonically swollen cells are less prone to hydroperoxide-induced LDH release than hypertonically shrunken cells, thus suggesting that cell swelling may exert beneficial effects during early stages of oxidative cell injury probably due to swelling-induced alterations in hepatic metabolism.

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver ofClarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role inC. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.

The roles of various inorganic ions and taurine, an organic osmolyte, in cell volume regulation were investigated in the perfused liver of a freshwater air-breathing catfishClarias batrachus under aniso-osmotic conditions. There was a transient increase and decrease of liver cell volume following hypotonic (-80 mOsmol/l) and hypertonic (+80 mOsmol/l) exposures, respectively, which gradually decreased/increased near to the control level due to release/ uptake of water within a period of 25–30 min. Liver volume decrease was accompanied by enhanced efflux of K⁺ (9.45 ± 0.54 µmol/g liver) due to activation of Ba²⁺- and quinidine-sensitive K⁺ channel, and to a lesser extent due to enhanced efflux of Cl^- (4.35 ± 0.25 µmol/g liver) and Na⁺ (3.68 ± 0.37 µmol/g liver). Conversely, upon hypertonic exposure, there was amiloride- and ouabain-sensitive uptake of K⁺(9.78 ± 0.65 µmol/g liver), and also Cl^- (3.72 ± 0.25 µmol/g liver). The alkalization/acidification of the liver effluents under hypo-/hypertonicity was mainly due to movement of various ions during volume regulatory processes. Taurine, an important organic osmolyte, appears also to play a very important role in hepatocyte cell volume regulation in the walking catfish as evidenced by the fact that hypo- and hyper-osmolarity caused transient efflux (5.68 ± 0.38 µmol/g liver) and uptake (6.38 ± 0.45 µmol/g liver) of taurine, respectively. The taurine efflux was sensitive to 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS, an anion channel blocker), but the uptake was insensitive to DIDS, thus indicating that the release and uptake of taurine during volume regulatory processes are unidirectional. Although the liver of walking catfish possesses the RVD and RVI mechanisms, it is to be noted that liver cells remain partly swollen and shrunken during anisotonic exposures, thereby possibly causing various volume-sensitive metabolic changes in the liver as reported earlier.