Cell volume regulation in the perfused liver of a freshwater air-breathing cat fish Clarias batrachus under aniso-osmotic conditions: roles of inorganic ions and taurine.

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 catfish Clarias 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 micromol/g liver) due to activation of Ba(2+)- and quinidine-sensitive K(+) channel, and to a lesser extent due to enhanced efflux of Cl(-) (4.35+/- 0.25 micromol/g liver) and Na+ (3.68+/- 0.37 micromol/g liver). Conversely, upon hypertonic exposure, there was amiloride-and ouabain-sensitive uptake of K+ (9.78+/- 0.65 micromol/g liver), and also Cl(-) (3.72 +/- 0.25 micromol/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 micromol/g liver) and uptake (6.38 +/- 0.45 micromol/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.

Cell volume changes affect gluconeogenesis in the perfused liver of the catfish Clarias batrachus.

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver of Clarias 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 in C. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.

Effects of anisotonicity on pentose-phosphate pathway, oxidized glutathione release and t-butylhydroperoxide-induced oxidative stress in the perfused liver of air-breathing catfish, Clarias batrachus.

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 of 14CO2 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 of Clarias batrachus. Conversely, hypertonic exposure (345 mOsmol/l) caused significant decrease of 14CO2 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 on 14CO2 production from [1-14C]glucose and biliary GSSG release were also observed in the presence of t-butylhydroperoxide (50 mmol/l). 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.

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.